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Università di Pisa
Scuola di Dottorato in Scienze Agrarie e Veterinarie
Programma di Medicina Veterinaria
Effects of seminal plasma on cryopreserved semen and
artificial insemination in the Amiata donkey (Equus Asinus)
Candidate: Chiara Sabatini
Tutor: Dott. Alessandra Rota
SSD: VET/10
2011-2013
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SUMMARY
ABSTRACT... 3
ARTIFICIAL INSEMINATION IN EQUIDS ... 4
Brief history of AI in the horse ... 4
Current use of AI with preserved semen in the horse ... 5
Current use of AI with preserved semen in endagered equid species ... 6
EQUINE SEMEN PRESERVATION ... 9
SEMEN PROCESSING FOR FREEZING ... 10
Cold-shock ... 10
Extenders ... 11
Ice crystals formation and the “solution effect” ... 12
Cryoprotectants ... 12
CAUSES OF REDUCED FERTILITY WITH EQUINE FROZEN SEMEN... 14
Cryopreservation induced damage ... 14
Post-mating endometritis ... 19
Seminal plasma removal ... 21
DONKEY SEMEN CRYOPRESERVATION ... 31
SEMEN PROCESSING FOR FREEZING ... 31
Donkey semen quality post-thaw ... 34
Causes of reduced fertility with donkey frozen semen ... 35
The cryoprotectant ... 35
The female ... 36
The removal of seminal plasma ... 38
RESEARCH ACTIVITY ... 43
STUDY ONE ... 43
STUDY TWO ... 50
STUDY THREE ... 55
STUDY FOUR - PART A ... 64
STUDY FOUR - PART B ... 66
GENERAL DISCUSSION ... 69
REFERENCES ... 77
ACTIVITIES DURING THE PHD STUDIES AND PUBLICATIONS ... 100
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ABSTRACT
Pregnancy rates in donkeys after artificial insemination (AI) with cryopreserved semen are still low, compared to the horse species. The aims of this thesis were to evaluate if the resuspension of frozen-thawed donkey spermatozoa in seminal plasma (SP) could affect semen quality and jennies endometrial response and fertility after AI. In Study 1 it was observed that the proportion of polymorphonucler cells (PMN) in uterine flushings performed 6-10 hours after AI was increased when 70% SP was added to the AI dose, compared to an extender. Moreover, seminal plasma showed a trend toward improvement of pregnancy rates but differences (60% and 20% for addition of SP and extender, respectively) were not statistically significant. In Study 2 it was shown how, compared to control (0% SP), 70% SP significantly decreased post-thaw motility and plasma membrane integrity during 2 hours of in vitro preservation at 37°C. In Study 3, where lower seminal plasma concentrations were tested (5 and 20%) during 4 hours of incubation at 37°C, sperm motility and the proportion of chromatin damaged sperm cells were not affected by the presence of SP immediately post-thaw, compared to control, while plasma membrane integrity was lower in the 20% samples. After 4 hours, however, all main semen characteristics were significantly worse in the 20% seminal plasma samples, compared to control, while the 5% SP samples showed intermediate values. In Study 4 no differences between AIs with 70% SP added or AIs without post-thaw dilution were seen for proportion of PMN evaluated in uterine flushings 24 hours after AI or fertility (1/5 in both groups). Combining the results of Studies 1 and 4, post-thaw addition of 70% seminal plasma to frozen-thawed donkey spermatozoa did not improve pregnancy rates, although there was a trend in this direction. In vitro, both 70% or lower SP concentrations (5% and 20%) did not improve neither motility nor viability or chromatin integrity. The presence of seminal plasma during AI promoted the PMN influx into the uterus 6-10 hours post AI and did not shorten mating induced endometritis compared to the control. In the donkey species, both the interactions of seminal plasma with the sperm cell and the endometrium and their effects on fertility still need to be better explained.
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ARTIFICIAL INSEMINATION IN EQUIDS
Brief history of AI in the horse
From the earlier report available on the use of the artificial insemination (AI) we know that the horse is apparently the first species in which this procedure was performed. In his detailed review of AI and semen preservation Watson (1990) reports the content of a book dated back to the year 700 of the Egira (1322 AD) that documents the successful attempt of a mare's owner to “steal” semen from a neighbor's valuable stallion and introduce it into the vagina of his mare thereby obtaining a foal. Whether this story is real or not, scientific research on AI was commenced 300 years later by the inventor of the microscope, Antonie Van Leeuwenhoek, who demonstrated that semen contains motile cells. Later on, in 1780, the Italian abbot Lazzaro Spallanzani successfully performed AI in dogs and also demonstrated that human, stallion and frog spermatozoa could recover their motility after being cooled in snow and subsequently rewarmed. Use of AI for commercial purpose in the horse began at the end of the 1800 in both America and Europe (Heape, 1897; Chelcowsky, 1894). At that time the common AI practice consisted in the recovery of semen from the vagina after mating and its subsequent introduction in the uterus of the mare by a syringe (Ivanoff, 1907). The first artificial vagina (AV) for semen collection was developed for the dog in 1914 while the first model of open-ended AV for stallions, called “Krakow” model, was produced in Poland (Bielansky et al, 1975). It consisted of a simple metal open style frame surrounded by two latex rubber liners (Allen, 2005) which is still the basic structure of one of the two most popular types of stallion's AV used today. Before World War 1 sporadic AI trials were performed across the world (reviewed by Aurich, 2012) but were not accompanied by a systematic research. Even if the outcome and the details of their experiences are poorly documented too, it appears that Russia and China where the first countries in which equine AI was employed massively: by 1938 approximately 120.000 mares had been artificially inseminated in Russia. Concurrently the first procedures for semen dilution and preservation were developed and Walton (1938) obtained pregnancies after AI with semen extended in a buffered glucose solution and stored 48 hours at 4°C. Moreover, the alteration of the sperm plasma membrane named “cold shock” was described and further studies allowed the discovery of the protective properties of the egg yolk during cooling (Phillips, 1939). Later on,
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preliminary and unsuccessful attempts to freeze semen were performed until Polge et al (1949) finally discovered the properties of glycerol as a cryoprotectant. At the end of the 50' the first pregnancy from frozen stallion spermatozoa was reported in Guelph, Ontario (Barker and Gandier, 1957). The Russian government, similarly to other countries, in the beginning funded AI interventions in order to provide horses for the military sector. However, despite of the advances achieved on semen manipulation and preservation, horses were later only seen as agricultural force and thus considered not enough valuable to justify a widespread use of AI.
Current use of AI with preserved semen in the horse
The equine breeding industry in Europe started to rely upon artificial insemination in 1960s-1970s concurrently to the great increase and diversity of equine sports. With the exception of the Thoroughbred stud book which still bands the use of AI, all others breed registries in Europe allow the use and shipment of both cooled and frozen semen. To give some examples of the European scenary, France and Germany experienced a five folds increase in the number of sport horse mares bred by AI from 1985 to 1995, ending up with more than 50% mares bred artificially. In 2004 this value almost reached 90%. In the United States, the American Quarter Horse Association (which is the largest breed registry in the country), approved in 1997 and 2001 the use of both cooled shipped and of frozen-thawed semen, respectively.
Some of the advantages for which AI and semen preservation (reviewed by Watson, 1990) became more and more common include: control of venereal diseases, removal of geographical limitations and availability of superior stallions across the world through the shipment of insemination doses instead of live animals, removal of temporal limitations since frozen semen can be stored indefinitely without loss of its fertility, availability of AI doses from stallions that are injured or dead if semen from these animals was previously frozen. Of course AI, particularly when frozen semen is used, also present some disadvantages (Watson, 1990; Loomis et al, 2011): both cooled and frozen semen are less fertile than fresh semen, veterinarians and skilled operators are required for proper semen processing, stallions differ in semen quality after cooling and freezing, cooled and especially frozen semen have lower longevity than fresh semen thus careful monitoring of the mare is needed in order to perform AI as close as possible to the ovulation. Frozen semen related issues will
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Current use of AI with preserved semen in endagered equid species
The genus equus includes three different subgenera: Equus (wild and domestic horses), Asinus (donkeys and wild asses), Dolichohippus and Hippotigris (Grevy's and Plain's zebra, respectively). Most wild equids are currently endangered and some of them were extinct and survived only after breeding in captivity like for example the Przewalski's horse (extinct in the wild in 1966 and later reintroduced in Mongolia and China; Souris et al, 2007). Donkeys and wild horses were originally domesticated and originated several local breeds that were used for the work in the fields and as pack animals. However, the increasing mechanization in countries and in towns during the XX century led to a disuse of these animals and the number of individuals for each breed soon declined (Smits et al, 2012). Several national organizations were born in order to monitor this phenomenon locally like for example the “Italian association for endangered native breeds” (R.A.R.E, www.associazionerare.it). According to the Food and Animal Organization (Scherf, 2000), in Italy 4 donkey breeds are extinct and 6 are critical or endangered while 2 horse breeds are extinct and 11 are critical or endangered. This classification accounts for breeds that include more than 100 and less than or equal to 1000 breeding females or the total number of breeding males is less than or equal to 20 and greater than 5. My thesis focuses in particular on the Amiata donkey breed which originates from Tuscany and is comprised in the above mentioned list. With respect of data collected in the year 2000 there were 62 herds remaining with 149 females registered in the herd book while five years later the registered individuals were 373 overall with 198 mares and 24 sires (Scherf, 2000; Kugler et al, 2007). However data from a local source in Tuscany states that today the overall Amiata donkey population consists of 1400 individuals (data available on www.filieraippicatoscana.it/cavalli/razze/36-asino-amiata).
Common strategies undertaken in order to preserve endangered animal breeds include a breed structure analysis, DNA profiling, accurate breeding programs and the constitution of a genome bank in which semen, embryos, oocytes and somatic cells can be deposited (Alderson, 2005).
The current status of the Amiata donkey preservation is so far strongly based on the use of technologies of reproduction as they were developed in the horse species. This also occurs for other endangered donkey breeds like the Poitou jackass in France (Trimeche et al, 1998).
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Beside the use of reproductive technologies for the preservation of endangered breeds, some countries around the world still express a great need of donkeys and mules as they are ideal animals for agricultural work in semi-arid regions. The Geographic and Statistics Institute of Brasil reported that in 2006 donkey breeding in the Northeast Brazil accounted for almost 91% of the national herd (IBGE, 2006). For these reasons during the last decade great effort was done by several Brazilian research groups in order to apply equine technologies of reproduction on this species and collect fertility data. The table below shows per cycle pregnancy rates obtained by cooled and frozen semen in horses and donkey (Table 1), Fertility of fresh semen AI in the donkey was studied as well and ranged from 51% to 73% (Marianelli et al, 2009; Oliveira et al, 2012).
Table 1: Per cycle pregnancy rates (PR) resulting from AIs with cooled and frozen semen in the donkey and the horse species
Cooled-preserved semen
Donkey Horse
Alvarez et al, 2004 26% 62-87.5% Metcalf, 1998.
Vidament et al, 2009 45-64% 39% Manning et al, 1998
Perulli, 2012 (unpublished) 41.7% 50% Burns et al, 2000 59.4% Loomis et al, 2001 R Raannggee::2266--6644%% RRaannggee::3399--8877..55%% Frozen semen
Trimeche et al, 1998 0-38% 58% Samper, 1995
Oliveira et al, 2006 0% 38% Barbacini et al, 1999
Vidament et al, 2009 0-11% 54% Vidament et al, 2000
Oliveira et al, 2012 28.3% 51.3% Loomis et al, 2001
R
Raannggee::00--3388%% RRaannggee::3388--5588%%
It is clear that fertility results for both cooled and frozen semen AI are lower in the donkey than in the horse. Unfortunately it is sometimes hard to make comparisons, since for example fertility of donkey cooled semen resulted in most cases from trials in which ejaculates were preserved for less than 12 hours (Alvarez et al, 2004; Vidament et al, 2009).
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More exhaustive results but yet unpublished are presented by Perulli (2012) who collected field data over 9 consecutive breeding seasons during which jennies were bred with semen that had been cooled shipped and used in the field about 24 hours later.
With respect to cryopreserved semen, the lower fertility in the donkey compared to the horse is more evident, in fact, pregnancy rates repeatedly resulted in 0% and ranged 0-38% vs 38-58% in the two species. The studies mentioned in Table 1 include a diversity of treatments applied to donkey spermatozoa either prior to cryopreservation or post-thaw that have been transposed on this species as they were developed for the horse. For this reason before discussing frozen semen related issues in the donkey, a review on semen cryopreservation in the horse will be given.
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EQUINE SEMEN PRESERVATION
Fertility in mares is reduced when cryopreserved semen is used compared to cooled semen (Loomis et al, 2001). Post-thaw semen quality is influenced by the freezing process itself but also by the species and individual differences. In contrast with bulls, which are farm animals and are selected also for their semen quality, stallions are selected for their morphology, sport performances or affection. This results in a great variation in semen quality and freezability between individuals, which in turn determine a great variation in fertility. Amann and Pickett (1987) analyzed semen from 40 stallions and observed that only 38% of them had post thaw motility that was similar to their pre-freeze values. All the other stallions experienced a decrease in motility of 65%. Vidament et al (1997) defined as freezability of a given stallion the proportion of ejaculates showing at least 35% progressive motility post-thaw over the total number of the ejaculates frozen. They found that, among 48 stallions evaluated freezability was 0%, 1-33%, 33-66% and >66% in 11, 7, 12 and 18 males respectively. In this study, fertility tended to be lower for stallions with freezability <33% but no correlation with per cycle fertility was observed, may be because AI doses were obtained from selected ejaculates only. In another study, Samper et al (1991) cryopreserved ejaculates from 9 fertile stallions and inseminated 177 fertile mares within 12 hours of ovulation with 109 spermatozoa, regardless of post-thaw quality. They found that first cycle and seasonal pregnancy rates ranged 32-70% and 60-90%, respectively. This study stresses out the importance of a careful management of the mare when she has to be bred with frozen semen but at the same time shows how, even when this goal is achieved, individual variability between stallions remain. To give a few more numbers and highlight differences between species, bull frozen semen fertility range between 51 and 64% (Phillips et al, 2004; Zhang et al, 1999) and is similar to fertility with fresh semen. In one study, per cycle pregnancy rate with fresh and frozen insemination doses containing the same sperm number and obtained from the same bull's ejaculates were 51.5% vs. 50.4% respectively (Bucher et al, 2009).
The efficiency of frozen-thawed semen is influenced by the species, the individual and the need to account for sperm losses caused by cryopreservation itself, thus it affects the number of sperm cells that are required in an insemination dose in order to maximize its fertility potential. Conventional frozen semen AI doses contain 300–500x106 spermatozoa for
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the horse (Allen, 2005), which is much higher than 20x106 spermatozoa used in the bovine industry (Watson, 1990).
Semen processing for freezing
Several freezing protocols are available for stallion semen but as a general principle a two step dilution is to be performed. After collection and evaluation, the ejaculate is usually extended with a skim milk based extender at a concentration of approximately 50x106 spermatozoa/mL and centrifuged for 10-15 min at 350-700g. Subsequently, the supernatant is discarded so that not more than 0-5% of seminal plasma is left (Amann and Pickett, 1987) and the sperm pellet is resuspended up to the desired concentration (usually between 100-200x106 spermatozoa/mL) in a freezing extender, either egg yolk or skim milk based, which is supplied with cryoprotectant agents (CPA). Extended semen can then be either cooled from room temperature to 4°C at a rate of 0.05°C/min and packed in 0.5 mL plastic straws, or packed in straws at room temperature and cooled right after. Straws are frozen by liquid nitrogen vapors (either in a programmable freezer or by suspending them a few cm above liquid nitrogen) from 4°C to a final temperature of -140°C using rapid rates (-15/-60°C/min) (Watson et al, 2000). Frozen straws are finally stored into liquid nitrogen (-196°C). As a consequence of the individual variability in semen freezability between stallions, several authors stress the importance of adopting a “split-ejaculate” test freeze in order to find which extender, cooling curve and thawing curve are the most effective for each animal (Loomis and Graham, 2008).
During the progressive decrease of temperature, a series of critical points in which sperm cells are damaged were identified and will be now presented together with the actions commonly undertaken to minimize their effects.
Cold-shock
This is the first stressful event that spermatozoa are exposed to while the temperature falls from body temperature to 0°C (expecially between 19 and 8°C; Moran et al, 1992; Kayser et al, 1992). As a response to cooling, the lipids of the plasma membrane pass from a liquid to a gel state and separate, causing a rearrangement of the intramembrane proteins too (Watson et al, 2000). Consequences of the cold shock for the sperm cells are abnormal patterns
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(circular or backwards) and rapid loss of motility, acrosomal damage, reduced metabolism and loss of intracellular components due to increased permeability of the plasma membrane (Amann and Pickett, 1987; Moran et al, 1992). The sensitivity of spermatozoa to the cold shock depends on the structure of the plasma membrane, which is species specific, with particular reference to both the phospholipid/cholesterol and the poliunsaturated/saturated fatty acids ratios. It was observed that a major content of cholesterol and saturated fatty acids in the plasma membrane as it is in the man and in the rabbit, increase the resistance of the sperm to the cold-shock. The boar is the most sensitive since its cholesterol to phospholids ratio is very low and the cholesterol is distributed asymmetrically (Johnson et al, 2000) while the ungulates are intermediate. Among ungulates, the sperm plasma membrane in the stallion has a lower ratio of cholesterol to phospholipids than in the bull (0.36 and 0.45, respectively; Parks and Lynch, 1992) the second being thus more resistant. The cold-shock related damage can be reduced by controlling the cooling rate (curves faster than -0.3°C/min should not be used; Aurich, 2005) and by adding protective substances to semen.
Extenders
The most common extenders contain either skim milk or egg yolk as protective agents against cold-shock, and act mainly by dilution or replacement of the seminal plasma, control of pH and osmolarity and energy supply to the sperm cells. Both skim milk and egg yolk, however, have a complex composition which makes it hard to standardize the production of the extenders and are susceptible to sanitary issues. Moreover, certain fractions may be beneficial to sperm functions during cooling while others may be detrimental. Fractionation of milk and subsequent studies on the effects of its purified proteins have led to the identification of phosphocaseinate and β-lactoglobuline as the most effective for supporting the sperm metabolism. These two proteins were subsequently included in one of the most used commercial extenders for cooled stallion semen (INRA96®, IMV Technologies, France) (Battelier et al, 1998) together with other substances that support the sperm cells like buffers, sugars and antimicrobials (Watson, 1990).
The components in the egg yolk which are responsible for protection of the sperm membranes during cooling are the low density lipoproteins (LDL) (Kampschmidt et al, 1953). It is not clear by which mechanisms this occurs in the stallion but studies conducted in the
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bull showed that LDL bind to lipid-binding seminal plasma proteins which would otherwise remove cholesterol from the sperm plasma membrane (Bergeron et al, 2004). A second hypothesis is that LDL associate with the sperm membrane and provide protection to the cell by stabilizing it (Watson et al, 1975). Since post-thaw semen quality did not differ between spermatozoa frozen in extenders containing either whole egg yolk or egg yolk plasma alone, the latter is preferred in order to overcome the sanitary and practical issues mentioned above (Pillet et al, 2011). Skim milk and egg yolk based extenders need to be added of permeating cryoprotectants (CPA) in order to be suitable for semen freezing but it is important to note that the sugars contained in skim milk and the LDL component of the egg yolk also act as non permeating CPA themselves.
Ice crystals formation and the “solution effect”
The second crucial step of the freezing process occurs when the temperature decreases until the freezing point (-5/-10°C) and pure water around the spermatozoa start to crystallize. As a consequence the amount of solutes around the cells increase and create a hyperosmotic environment. While the water starts to leave the sperm cytoplasm and move to the extracellular space in order to restore the osmotic balance, the cells progressively shrink. This phenomenon is called “solution effect” (Mazur et al, 1970) and has potentially lethal consequences so its duration should be minimized by choosing a cooling curve which is not too slow. At the same time, the rate of cooling should not be so rapid that the intracellular water cannot leave the cytoplasm because intracellular ice crystals would form and rupture the membrane. After slow cooling to 4°C, stallion spermatozoa are usually frozen at a rate comprised between 20 and 100°C/min (Sieme et al, 2008) down to -196°C (Palmer, 1984). The thawing curve is important as well because slow rewarming causes the phenomenon of “recrystallization” which is of course harmful. Conventionally, equine semen frozen in 0.5 ml straws is thawed by immersion in a waterbath at 37°C for 30 seconds (Watson, 1990)
Cryoprotectants
As already said, Polge et al (1949) discovered that glycerol (GLY) was necessary for sperm survival during freezing. Since that, several CPA were identified and tested and then classified as permeating and non permeating. By penetrating the cell, CPA such as glycerol, dimethyl
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formamide (DMF), DMSO and ethylene glycol (EG) (Watson, 1990) reduce the osmotic imbalance between intra and extracellular space and increase the amount of water left unfrozen so that the solutes around the sperm cell are more diluted. Both these two actions depress the freezing point and reduce the solution effect thus protecting spermatozoa during freezing. Unfortunately all permeating CPA can be toxic to spermatozoa in a dose dependent way. Glycerol, which is most commonly used, is incorporated in freezing extenders for stallion semen at a concentration comprised between 2 and 5% (Loomis et al, 2008). Recent studies showed that this CPA damaged the sperm membrane when present in concentrations above 3.5% while it had toxicity upon the cytoskeleton by inducing depolimerization of the F actin already at a concentration of 1.5% (Marcias Garcia et al, 2012). Moreover glycerol has a high molecular weight which delays his entrance into the sperm cytoplasm compared to other permeating CPA. For these reasons, the efficacy of other permeating CPA at different concentrations was evaluated. One study showed that methyl formamide, DMF and EG at either 0.3, 0.6 or 0.9M concentration provided post-thaw total and progressive motility similar to glycerol at 0.55M (Squires et al, 2004). Vidament et al (2002) observed that a combination of 1 or 3% DMF with 1 or 3% GLY gave higher post-thaw sperm motility than DMF or GLY alone. Moreover they obtained similar pregnancy rates from mares inseminated with spermatozoa frozen in extender containing 2% glycerol, 3% glycerol, and 2% DMF (PR 46%, 58% and 50%, respectively). With respect of EG, one study showed no difference between post thaw motility and membrane integrity of sperm cells frozen using a lactose-EDTA-egg yolk extender with 3.5% EG compared to a lactose-lactose-EDTA-egg yolk extender with methyl cellulose, trehalose and acetamide. However, AI were performed only with the latter treatment and a conception rate of 23% was obtained (Snoeck et al, 2012). No recent studies are available on the effects of EG on fertility of stallion frozen semen, in one study a 50% PR with semen frozen in presence of 6% EG was observed but only 6 mares were inseminated (Kotjagina et al, 1963). Hoffman et al (2011) cryopreserved semen from stallions classified as “good” and “bad” freezers according to Vidament et al (1997) in INRA-82 + 2% egg yolk supplemented with 1%, 2%, 3% or 4% of GLY, EG, methyl formamide, or DMF. They found that concentrations ranging from 2 to 4% gave the highest post thaw motility values for all CPA but “bad freezers” always showed lower post-thaw motility values than “good freezers”. While sperm chromatin integrity was affected neither by the CPA nor by its concentration, sperm viability and acrosome integrity were higher with GLY. Interestingly,
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DNA quality started decreasing when CPA concentration was greater than 2% for “bad freezer” stallions, while “good freezers” could tolerate concentrations up to 4%.
Non permeating CPA include a variety of sugars like glucose, lactose, sucrose, raffinose, trehalose and other compounds like polyvinyl pirrolidone, glicine and glutamine. These are thought to act by increasing the amount of unfrozen water, reducing the concentration of salts or directly interacting with lipid and proteins of the sperm membrane (as it is the case of trehalose and glycine; Rudolph et al, 1986) but are only effective when used in combination with permeating CPA (Holt, 2000).
Causes of reduced fertility with equine frozen semen
Despite the advancements done in semen cryopreservation technologies over time, the adoption of innovative semen processing techniques prior to freezing and the customization of freezing protocols for each stallion, fertility of frozen semen in the horse remain low, most often requiring at least two inseminations to obtain a pregnancy (Table 1). Watson (1990) reported that 40-50% of the spermatozoa in a semen sample do not survive cryopreservation and that the survivors exhibit more or less severe modifications that reduce their lifespan. However, the causes of reduced fertility with frozen semen are not limited to post-thaw sperm damage alone and include factors related to the mare and to the removal of seminal plasma before cryopreservation.
Cryopreservation induced damage 1) “capacitation like changes”
In order to acquire the ability to fertilize an oocyte, ejaculated spermatozoa need to undergo through the steps of capacitation which is triggered by the biochemical characteristics of both the seminal fluid and the female tract. HCO3- concentrations are increased at these levels and this molecule enters the sperm membrane through a HCO3-/Na2+ dependent transport. HCO3- increases cyclic adenosine monophosphate (cAMP) metabolism whose main target is the protein kinase A (PKA) that is so activated. PKA is responsible for the tyrosine phosphorylation of several proteins that can thus initiate extracellular signals transduction, intracellular transports and cell cycle progression involved in capacitation. Some of the most important changes of sperm capacitation occur at the level of the plasma
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membrane. Sperm membrane is a phospholipid bilayer with sphingomyeline (SM) and phosphatidylcholine (PC) being present in the outer leaflet and phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the inner leaflet. HCO3- induces phospholipids “scramblase” with PE and PS appearing on the outer leaflet. The major effects of scrambling are to enable cholesterol efflux from the plasma membrane which is also mediated by albumin (a cholesterol acceptor) and to redistribute some membrane proteins. As a consequence, membrane fluidity is modified and surface receptors that will increase sperm-oocyte affinity are exposed. The extracellular ion concentrations in the female tract modifies the intracellular concentration of these ions in the sperm cell leading to alteration of the membrane potential. Sperm plasma membrane thus become hyperpolarized (negative ions are increased compared to the extracellular environment) and low voltage Ca2+ channels are activated thereby increasing the influx of Ca2+ in the cell. Increased intracellular cAMP and Ca2+ are associated with the expression of hyperactived motility, in fact Ca2+ is responsible for increased flagellar asymmetry which characterize the asymmetric non progressive pathway of hyperactivated motility, while cAMP is responsible for initiation and mainteinance of flagellar beating (for review, Suarez and Ho, 2003; Gadella and Visconti, 2006). This form of motility is thought to aid the sperm progression through the oviduct and the penetration of the zona pellucida. Soon after binding to the ZP, capacitated spermatozoa undergo the acrosome reaction i.e. the fusion of the outer acrosomal membrane with the sperm plasma membrane that allows the content of the acrosome, mainly enzymes, to be released outside the sperm cell. Acrosomal enzymes are glycohydrolases, proteinases, esterases, sulfatases, phosphatases and phospholipases (Tulsiani et al, 1998) that digest the cumulus oocyte complex making a hole through which spermatozoa can pass and fuse with the oocyte. As described earlier, cryopreservation may induce a structural reorganization or modifications of the sperm plasma membrane. Dobrinski et al (1995) showed that these changes can impact the ability of the sperm cell to interact normally with cells of the femal tract: fewer cryopreserved compared to fresh spermatozoa were able to attach to oviductal epithelial cells in vitro. Moreover, cryopreserved spermatozoa were less able to bind to homologous ZP compared to spermatozoa stored at room temperature. Watson (1995) suggested that cryopreserved spermatozoa are in a partially capacitated state possibly as a consequence of increasing concentrations of Ca2+ that enter the sperm cell because of the cooling/freezing induced membrane permeability.
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Recently, Thomas et al (2006) observed higher phospholipid scrambling (particularly with respect of phosphatidilserine externalization) and more proteins displaying tyrosine phosphorilation in frozen-thawed spermatozoa compared to sperm cells either fresh or incubated at room temperature. However, inhibition of the PKA-mediated protein tyrosine phosphorylation in cryopreserved cells did not reduce scrambling as instead did in in vitro capacitated cells used as control. Moreover, patterns of protein tyrosine phosphorilation in these two groups were different and when acrosomal reaction was induced a higher proportion of in vitro capacitated versus cryopreserved cells underwent the process. These findings suggest that cellular modification induced by cryopreservation are produced differently than in physiologic capacitation, even if the result looks similar.
2) Apoptosis-like changes
In the absence of fertilization spermatozoa are physiologically destined to a programmed senescence which resembles the intrinsic pathway of apoptosis in somatic cells. Moreover, germ cells exhibiting apoptosis were detected in stallion testicular parenchyma particularly at stages IV-VI of meiosis and at mitotic proliferation of B1 and B2 spermatogonia. Apoptosis at this level is thought to regulate germ cell numbers and eliminate the defective ones during spermatogenesis (Heninger et al, 2004).
The intrinsic pathway of apoptosis in somatic cells is activated in presence of a stressor (oxidative stress, heat, hypoxia, DNA damage or lack of prosurvival factors) that disrupts the cellular homeostasis and the equilibrium between anti and pro apoptotic factors. Proapoptotic members are thus activated by proapoptotic proteins and, as a consequence, cytochrome c and other apoptogenic factors are released from the mithocondria, leading to formation of the apoptosome, which activates caspases 9 and subsequent activates the effectors caspases 3 and 7 (Rasola and Bernardi, 2007).
In spermatozoa, the main anti apoptotic factor appear to be phosphatidylinositide 3-kinase (PI3K) while the negative regulator of the system is the phosphatase and tensin homolog PTEN which is located in a different compartment of the cell so that in optimal conditions it does not interfere with PI3K. The activity of PI3K consists in binding to AKT1 (serine/threonine kinase) and maintaining it in a phosphorilated status which is necessary to promote sperm cell survival. In human spermatozoa it was observed that if PI3K is inhibited spermatozoa default to the intrinsic apoptotic cascade and display rapid motility loss,
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mithocondrial ROS (reactive oxygen species) generation, caspase activation in the cytosol, phosphatidylserine externalization, cytoplasmic vacuolization and oxidative DNA damage (Koppers et al, 2011).
Apoptosis like changes were detected in stallion cryopreserved spermatozoa and consisted of increased membrane permeability, low mithocondrial membrane potential and caspase activation (Ortega-Ferrusola et al, 2008; Brum et al, 2008). Caspase 3, 7 and 9 were identified in fresh, cooled and frozen equine spermatozoa (Ortega-Ferrusola et al, 2008). It is thought that the mechanism through which cryopreservation induces apoptosis is mainly linked to the oxidative stress caused by increased ROS production that is known to be a major consequence of freezing.
3) Oxidative damage
During cooling spermatozoa are subjected to oxidative damage that affects membrane phospholipids, proteins and chromatin (Ball, 2008). The reasons why spermatozoa are particularly susceptible to oxidative stress compared to other cells were resumed by Aitken et al (2014):
1) the sperm cell has very little intracytoplasmic space, mainly located in the midpiece, so only little amount of antioxidants can be hosted inside the cell. For this reason sperm antioxidant protection mostly rely on seminal plasma which was found to contain several enzymatic antioxidants. The activities of catalase (98.7 ± 29.2 U/mg protein) which is a scavenger for hydrogen peroxide (H2O2), superoxide dismutase
(SOD; 29.15 ± 6.64 U/mg protein) which is a scavenger for superoxide anion (O2-) and
glutathione peroxidase (GPX; 0.87 ± 0.06μM NADPH oxidized/min/mg protein) which scavenges hydrogen peroxide and lipid peroxides, were determined in equine seminal plasma (Ball et al, 2000; Baumber and Ball, 2005). In addition to these compounds, low weight factors such as albumin, taurine, urate, pyruvate, lactate, ascorbic acid, tocopherol, ergothioneine may act as antioxidants (reviewed by Ball, 2008). Unfortunately, as it will be discussed later, seminal plasma needs to be almost entirely removed before semen cryopreservation (Moore et al, 2005) thus depriving spermatozoa of their main source of protection against oxidative stress.
2) spermatozoa can generate ROS from two main sources: a NADPH oxidase (NOX5) and the mithocondria. NOX5 is located on the sperm membrane and under physiologic
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conditions it promotes a lesser production of O2- (superoxide anion) which is involved
in sperm capacitation by promoting tyrosine phosphorylation. Mithocondria are the main source of energy for the sperm cell since they produce ATP by oxidative phosphorilation. In the mithocondrial electron transport chain, continuous leakage of electrons lead to formation of O2- which is normally dismutated in H2O2 and then
broken to water by mithocondrial SOD and GPX, respectively (Pena et al, 2011). The third possible cellular source of ROS is the aromatic L amino acid oxidase that, in presence of oxygen, catalyzes the deamination and dehydrogenation of an aromatic aminoacid (like phenylalanine which is contained in egg yolk) to form H2O2 and
ammonia (Tosic and Walton, 1950). Baumber et al (2000, 2003) observed that incubating spermatozoa with a X-XO system (xanthine-xanthine oxidase) in order to induce generation of both O2- and H2O2 experimentally, caused a significant decline in
sperm motility and increased DNA fragmentation. In a subsequent experiment neither the addition of SOD nor of glutathione could reduce the generation of H2O2 as
instead did catalase. Moreover, in those studies only catalase was able to prevent the decrease in motility parameters and to reduce DNA fragmentation. These results suggest that H2O2 and not O2- is the major responsible for sperm oxidative damage
which is in accordance with the less stable nature of O2- that rapidly dismutates in
H2O2 both spontaneously and enzymatically (SOD). The presence of cryodamaged,
non-viable or morphologically abnormal spermatozoa (particularly those cells carrying excess cytoplasm as is the case of proximal droplets) in a semen sample is thought to increase ROS production both because of increased electron leakage from the mithocondrial electron transport chain and also because of a higher cytoplasmic content of the enzyme glucose-6-phosphate dehydrogenase that elevates NADPH concentration thus promoting NOX-5 activity (Ball, 2008; Musset et al, 2012).
3) the sperm plasma membrane has a high content of PUFA (poly unsaturated fatty acids) that confer fluidity in order to facilitate sperm-oocyte fusion. However, due to their weak double bounds, PUFA are a preferential substrate for ROS attack. Lipid peroxidation starts with hydrogen abstraction by ROS and subsequent generation of carbon centered lipid radicals. These combine with oxygen to produce peroxyl (ROO.) and alkoxyl (RO.) radicals that, in order to stabilize, extract hydrogen atoms from carbons on adjacents lipids. This reaction creates additional lipid radicals that
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perpetuate lipoperoxidation whose end up products are cytotoxic aldehydes such as malondialdehyde and 4-hydroxynonenol. These lipid aldehydes can react with both succinate dehydrogenase, a constituent of the electron transport chain, thus stimulating additional ROS production by mithocondria (Aitken et al, 2014) but also with DNA thereby concurring to the increased chromatine damage caused by H2O2
itself (Baumber et al, 2003). Increased lipoperoxidation was observed both in cooled (Ball and Vo, 2002) and cryopreserved semen (Ortega-Ferrusola et al, 2009) compared to fresh semen in a stallion dependent manner, as detected by the fluorescent probe C11-BODIPY581/591 and it was more pronounced over the region of the sperm midpiece.
In the latter study, moreover, high negative correlations between the proportions of spermatozoa having LPO and intact membranes, and spermatozoa having high mithocondrial membrane potential and caspase activity were observed.
The above presented findings suggest that ROS production during cryopreservation is a self-perpetuating process which is interrelated with LPO, apoptosis and DNA damage. All these mechanisms together decrease sperm quality thereby reducing the lifespan and possibly the fertility of frozen-thawed semen.
Post-mating endometritis
PME is a physiologic and temporary inflammation that develops into the uterus as a response of the endometrium to the presence of spermatozoa. In the beginning it was thought that the only responsible for this reaction were the bacteria present in the semen but when a sterile suspension containing spermatozoa was infused into the uterus the same response was evoked (Kotilainen et al, 1994). Sperm cells behave like antigens in the female genital tract and originate innate immune reactions which involve release of inflammatory mediators including the complement system. Through its activation, particularly cleavage of factor C5 into C5a and C5b, the sperm cell promote a chemotactic signal to PMN which flow into the uterus as soon as 0.5 hours after AI and peak within 8 hours (Katila et al, 1995). The mechanism of PMN binding to spermatozoa is not fully understood but it was demonstrated that, in addition to a conventional ligand receptor binding promoted by complement-mediated opsonization of the sperm cells (Troedsson et al, 2006), the extrusion of nuclear DNA and histones from PMN form so called neutrophil extracellular traps (NETs) that
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entangle the sperm cells and whose formation is increased in a time dependent way (Alghamdi et al, 2005). After binding to PMN spermatozoa are phagocytized.
Moreover, activated PMNs secrete prostaglandins (PGF2a) that stimulate miometrial contractions together with PGF2a and oxytocin that are produced due to the mechanical stimulation of the vagina and the cervix during natural mating or AI. Uterine contractions are necessary to clear the uterus from excess spermatozoa, bacteria, other debris and inflammatory mediators in order to restore a clean environment prior to the descent of the embryo, which occurs within 5 to 6 days after ovulation (Betteridge et al, 1982). Uterine clearance should also be completed before increasing progesterone concentration determines the closure of the cervix, otherwise subsequent accumulation of intrauterine fluid in diestrus would stimulate prostaglandin production and cause embryonic loss due to premature lutelolysis (Lehrer, 1988). While most mares are able to spontaneously resolve PME within 36-48 hours (mares resistant to PME, RPME), some mares show delayed clearance and thus the physiologic inflammation becomes persistent (mares susceptible to PME, SPME). Impaired myometrial contractility appear to be a key factor in determining susceptibility to PME as demonstrated by electromyographic monitoring of the miometrium. Myometrial activity during artificially induced endometritis was similar in susceptible and resistant mares 6 hours after inoculation of S. Zooepidemicus but while contractility persisted up to 19 hours in resistant mares, susceptible mares came back to baseline levels (Troedsson et al, 1993).
Another study reported production of nitric oxide (NO), a smooth muscle relaxant, and expression of inducible nitric oxide sinthase (iNOS) in the endometrium during PME. Thirteen hours after breeding, NO concentration in uterine secretions were higher in SPME mares compared to RPME, suggesting that myometrial contractility in these mares could be less efficient. Moreover, a higher number of mastocytes evaluated on endometrial byopses were immunopositive for iNOS in SPME mares (Alghamdi et al, 2005).
Differences between RPME and SPME mares were also seen in the expression of some inflammatory cytokines involved in PME after breeding with freeze-killed spermatozoa. In both groups cytokines increased 2 hours after the challenge, peaked between 2 and 12 hours and then decreased. However, subtle variations in the rise toward the peak and the subsequent decline were seen, with RPME showing anticipated IFNγ (Interferon gamma) peak (at 2 hours compared to 6 hours in SPME) and TNFα (Tumor necrosis factor alfa)
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concentration that didn't differ in SPME over time while it rose from hour 2 to hour 6 and then declined in RPME. In conclusion, the authors observed that overall cytokines concentration in SPME tended to rise and decrease more slowly than in RPME, possibly prolonging the inflammation. Moreover, in the same study susceptible mares showed higher PMN number on endometrial byopses 2 and 12 hours after breeding compared to RPME (Woodward et al, 2013).
Beside individual differences in susceptibility to PME, a more pronounced endometritis is thought to be the female-related factor affecting fertility of frozen semen AI in the horse. Kotilainen et al (1994) observed higher PMN concentration when mares were bred with frozen semen (59±20 x 106 PMN/ml) compared to both raw (7.4±2.5 x 106 PMN/ml) or extended fresh semen (5.0±4.4 x 106 PMN/ml). In the same study, AI with 800x106 spermatozoa either frozen or concentrated fresh (2ml final volume for both) gave similar neutrophil concentration. The authors thus concluded that the high concentration of the insemination dose could explain the stronger endometrial inflammation observed after frozen semen AI. According to unpublished data reported by Troedsson et al (2001), PMNs number were reduced at 24h compared to 6 and 12h after AI with spermatozoa in presence of seminal plasma while no decrease during the first 24 h post AI was observed when sperm cells were deprived of seminal plasma and extended with a commercial extender. Since common procedures for semen cryopreservation include seminal plasma removal, the absence of this substance could increase PME duration after breeding with frozen semen, thereby negatively affecting the chances to obtain a pregnancy.
Seminal plasma removal
Seminal plasma is made up by the fluids of the cauda epididymis and the accessory sex glands. Presence and size of these glands varies between animal species thus influencing volume and composition of seminal plasma. As mentioned above, current semen cryopreservation guidelines recommend complete separation of spermatozoa from seminal plasma to be achieved in order to reduce the volume of the insemination dose and to replace the latter with extenders containing CPAs and other substances that can protect spermatozoa during cooling and freezing (Amann and Pickett, 1987). However, recent reports suggest that low SP concentrations during cryopreservation might actually be beneficial to sperm quality post-thaw. Katila et al (2002) observed that, in 2 out of 3 stallions, total motility, progressive
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motility and proportion of rapid spermatozoa post-thaw were improved by restoration of seminal plasma prior to freezing in a proportion of 20% (v/v) of the extended semen sample. Moore et al (2005) centrifuged ejaculates from 12 stallions through a Percoll gradient and subsequently resuspended the sperm pellets into a freezing extender containing 0, 5, 10, 20, 40 or 80% seminal plasma. Interestingly, no significant differences in sperm motility and proportion of viable-acrosome intact spermatozoa were observed post-thaw, however as SP concentration rose above 20% the evaluated parameters tended to decline. According to these studies it can be suggested that a concentration of seminal plasma comprised between 5 and 20% in frozen semen is not detrimental to its quality.
Seminal plasma composition
Constituents of stallion SP have been studied since long time ago (Amann et al, 1987) and have been reviewed in detail (Katila and Kareskoski, 2006; Rodriguez-Martinez et al, 2011; Topfer-Petersen et al, 2005) thus this paragraph is meant to give a brief overview on the most significant information.
a) Ions and trace elements. Pesch et al (2006) determined SP concentrations of Na+, Ca (mostly represented in his ionized form Ca2+), Mg, P Cl, Cu, Fe and Zn with Na+ and Cl being the most represented (110 and 114 mmol/L each). The concentration of both total and ionized calcium were found correlated to the ejaculate volume, thus the accessory sex glands are the main contributors to Ca in seminal plasma. Fe and Zn instead are negatively correlated with semen volume and positively related to sperm concentration thus probably originating from the testis or the epididymis. Individual differences in Ca, Mg and Cu concentrations were seen in a study that included 15 fertile stallions (Barrier-Battut et al, 2002).
b) Enzymes. In their study Pesch et al (2006) detected the activity of the following enzymes: aspartate amino tranferase (AST), g-glutamyl transferase (GGT), alkalyne phosphatase (ALP), acid phosphatase (AcP), lactate dehydrogenase (LDH) of which GGT and LDH were correlated with sperm motility. The levels of both ALP and AcP were correlated positively with the sperm count and negatively with semen volume. It was previously observed that as ALP activity is mainly derived from the testes and the epididymides, its measurement can be used to diagnose ejaculatory failure (Turner and McDonnell 2003). Other authors showed that SP contains several glycosidases that bind to spermatozoa and are thought to be
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involved in dispersion of the cumulus oocyte complex before fertilization (Rethinaswamy et al, 1994). Activity of other enzymes such as lipase, angiotensin I converting enzyme and platelet activating factor acetylhydrolase were detected in stallion SP (Ball et al, 2003; Hough and Parks, 1994).
c) Antioxidants. As already mentioned SOD, GPx and catalase are the enzymatic anti-oxidants in equine seminal plasma. Catalase mainly originates from the prostate, Gpx mainly originates from the testis and the epididymis while highest SOD-like activity was detected in the ampulla and prostate (Baumber and Ball, 2005). Moreover, low molecular weight components contribute to the antioxidant activity of seminal plasma, particularly albumin, urate, taurine, hypotaurine, pyruvate, lactate, ascorbic acid, tocopherol and ergothioneine (Ball, 2001).
d) Hormones. Oxytocin as well as prostaglandins (PGE2 and PGF2α) and estrogens were detected in equine seminal plasma and might be involved in sperm transport and elimination after mating or AI as well as in PME (Claus et al, 1992; Watson et al, 1999)
e) Carbohydrates and lipids. Sugars like glucose, fructose, galactose, mannose and fucose were found either free or bound into glycoproteins and glycopeptides, with galactose being the most represented (Amann et al, 1987). Minelli et al (1998) studied the activity of prostasomes, vesicles containing high concentrations of cholesterol and sphyngomielin that were shown to interact with the sperm membrane conferring endopeptidase activity to spermatozoa. Prostasomes are more represented in the sperm rich fraction of the ejaculate but their function remain unknown.
f) Proteins. They were originally named Horse Seminal Plasma proteins (HSP from 1 to 8) and their amount in stallion SP was evaluated as approximately 10mg/ml per ejaculate. These proteins are mostly low molecular weight (14-30kDa) and are found in aggregates. Through a combination of affinity-chromatography, high performance liquid chromatography, electrophoresis and amino acid sequencing, the structural characterization of these proteins was determined, identifying these major classes: Fn-2 type, cystein rich secretory protein (also called CRISPs), kallikrein like protein and spermadhesin. HSP-5 could not be related to known proteins. Most of the isolated proteins (HSP1 and 2, HSP5 to 8) were found to be associated to the sperm surface and show heparin binding ability. Since evidence was provided that heparin is involved in sperm capacitation, its presence suggest that SP proteins could be involved in the process. Although an involvement of SP protein in several steps of
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fertilization was suggested, so far the precise role of each one of these is unknown. CRISP3 was found to partecipate in mating induced endometritis, as it will be discussed later, while HSP-7 was shown to bind to intact equine zona pellucida and is thought to mediate the sperm-oocyte fusion (Reinert et al, 1996). Evaluation of SP composition in the different fractions of the stallion's ejaculate showed that, compared to the sperm-rich fraction, the pre-sperm fraction is poor in protein content and that HSP-1 and HSP-2 are the most represented proteins in all fractions.
Recently other proteins have been described, such as lactoferrin, transferrin (both iron binding proteins, the first being involved in PMN-spz binding, as will be discussed later), leptins and growth factors (reviewed by Topfer-Petersen et al, 2005).
Evaluation of seminal plasma activities in vitro
a) On sperm cells. The effects of low SP concentrations during sperm cryopreservation such as the contribution, still mostly undefined, of some SP components to fertilization has already been discussed. With respect of cryopreserved semen, some studies exist in which spermatozoa were resuspended in seminal plasma post-thaw in order to evaluate if their characteristics could be improved. When 20% of either homologous or autologous seminal plasma was added to sperm post-thaw, plasma membrane and acrosome integrity increased while progressive motility decreased, five minutes after dilution (De Andrade et al, 2011). Furthermore, in a similar experiment, protein tyrosine phosphorylation but not lipid peroxidation was reduced during two hours of incubation (De Andrade et al, 2012). More detailed information on these studies will be presented in the discussion of this thesis.
b) On sperm-PMN interaction. The evidence that cryopreserved semen, deprived of seminal plasma, results in a stronger PME after AI fueled intense research on the activity that SP might have against this inflammation. Alghamdi et al (2004) evaluated sperm-PMN binding during incubation in uterine secretions collected from five mares 12 hours after fresh semen AI. The incubation was performed in presence of either seminal plasma, semen extender or a mixture of the two. While initially no significant difference was observed between the treatments, after 1 hour and for all the duration of the experiment (4 hours) both SP and seminal plasma plus extender markedly reduced sperm-PMN binding. Similar results were obtained after incubation of spermatozoa with blood derived PMN in presence of seminal
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plasma or seminal plasma proteins (these were obtained from a SP pool by precipitation with ammonium sulfate). Moreover, in the same study an in vivo trial was performed in which normal mares where challenged with dead sperm AI in order to induce PME, followed 12 hours later by fresh semen AI with spermatozoa either suspended in extender or in seminal plasma. Interestingly, pregnancy rates obtained for the two treatments were 5% (1/22) and 77% (17/22), respectively, thus providing indirect evidence of a modulating activity of seminal plasma on PME in vivo. Later on, Troedsson et al (2005) aimed at understanding whether the protective effect of SP was specific to viable spermatozoa only or not. First they found that seminal plasma could reduce sperm-PMN binding by suppressing sperm opsonisation, subsequently they incubated separately viable and snap frozen spermatozoa with PMN in presence of SP or SP proteins and observed that for both treatments PMN binding to dead spermatozoa increased while it was decreased to viable sperm cells. Beside suppression of sperm opsonization, Alghamdi et al (2005) demonstrated that SP proteins have endonuclease activity. They first used plasmid DNA as a substrate and then evaluted this effect on NETs produced by live PMN incubated with spermatozoa or E. Coli, in comparison to purified DNAse I. Interestingly they observed that SP proteins were 50% more efficient than DNAse I in rarefying PMN NETs produced against spermatozoa, suggesting that additional mechanisms apart from DNAse activity are responsible in SP for this effect. Furthermore, since the NETs produced by PMN against E. Coli were not dissolved, it can be inferred that SP does not interfere with the bactericidal activity of PMN. Even if both Troedsson et al (2005) and Aghamdi et al (2005) did not identified yet specific proteins or protein complexes to which the observed results could be attributed, techniques of proteins separation and purification performed in the first study revealed that at least two separate proteins were involved in the modulation of sperm-PMN interaction. Recently, Troedsson et al (2010) and Doty et al (2011) processed a pool of SP from 5 stallions as follows: they precipitated SP protein with ammonium sulfate, determined which protein pools showed biological activity (inhibition of sperm-PMN binding), separated and purified proteins in the chosen pools and analyzed their structure. As confirmed also by western blotting the protein responsible of the effect of interest was identified as CRISP-3. Its properties were subsequently analyzed by flow cytometry in comparison to seminal plasma and PBS: a significant suppression of PMN-sperm binding by SP and CRISP3 compared to PBS was observed (22.08% ± 3.05%, 2.06% ± 2.02% and 63.09% ± 8.67%, respectively). Moreover, when live spermatozoa were selected and
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incubated with either SP, CRISP3 alone or a control media, the same result was obtained thus confirming that SP, particularly CRISP3, selectively protects viable spermatozoa from PMN attack. The most recent findings on the PME modulating activity exerted by seminal plasma highlighted the activity of another SP protein, lactoferrin. This protein is highly expressed in the corpus and cauda epididymis but not in the testis, and was found to bind to dead spermatozoa only (Alghamdi et al, 2014). After incubation of snap frozen spermatozoa with blood derived PMN in presence of seminal plasma or of lactoferrin, the binding of PMNs was significantly enhanced compared to a control media (Troedsson et al, 2014). These results suggest that lactoferrin might facilitate recognition of dead sperm cells during PME thus promoting PMN binding to them.
Biomarkers of semen quality, freezability and fertility in seminal plasma in vivo
As already discussed, individual differences between stallions contribute to determine the ability of their semen to sustain well cryopreservation or not. Unfortunately the origin of these differences is not completely understood yet and increasing efforts have been done toward the identification of fertility biomarkers in stallions ejaculates. Since equine seminal plasma proteins appeared to be involved in the normal sperm function and in the sperm-oocyte interaction, some authors attempted at determining if any association could be seen between these proteins and semen quality, freezability and/or fertility. Studies in which SP protein content was analyzed in relation to stallion's fertility after fresh semen AI came up to different results. Brandon et al (1998) observed a higher content of HSP-1 in stallions with higher pregnancy rates, and an overall positive relation between this protein and fertility. HSP-2 and 4 were instead more represented in stallions with lower pregnancy rates and overall negatively related to fertility. Novak et al (2010) found positive correlations between CRISP-3 and fertility while kallikrein-1E2, HSP-1 and HSP-2 showed negative correlations. The authors found a significant regression equation (R2=0.77, P<0.0001) to predict first cycle pregnancy rate that included clusterin, HSP1 and cytrate sinthase. Moreover, insuline like growht factor-1 (IGF1) content was negatively related with per cycle pregnancy rate. Macpherson et al (2002) instead observed that both sexual rested and active stallions with high IGF1 concentration had higher per cycle pregnancy rate with fresh semen, although for sexually active stallions the statistical significance was not achieved.
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that were previously classified as “good freezers”, having at least 30% post-thaw progressive motility on three consecutive frozen ejaculates, and “bad freezers” having ≤20% post-thaw progressive motility. Aliquots of individual ejaculates from these animals were exposed to either autologous SP or to SP of a random stallion with the opposite semen freezability at a concentration of 30%. After further suspension in a commercial extender and incubation for 10 minutes, the samples were centrifuged and almost all the supernatant removed in order to process the sperm pellets for freezing. Post-thaw motility in “bad freezers” was improved by exposure to “good freezers” SP (29% and 34.5% respectively) while the reverse effect on “good freezers” was less pronounced (32% and 30% post thaw motility for spz from “good freezers” extended with autologous and “bad freezers” SP, respectively). A beneficial effect of heterologous seminal plasma addition was also seen on proportion of membrane intact spermatozoa in “bad freezers”, that increased from 25% to 34%, while any significat difference was observed for “good freezers”. Unfortunately, seminal plasma composition in this study was not evaluated, thus the reasons of the improved resistance to cryopreservation of “bad freezers” semen could not be elucidated. Jobim et al (2011) used a 35% post-thaw progressive motility as a limit to define a frozen ejaculate as viable. Thus ten stallions included in their study were classified as “good” or “bad freezer” if they had more or less than 30% viable ejaculates respectively. After evaluation of the respective content of SP proteins, they found that CRISP-3 and HSP-2 were more represented in “good freezers” ejaculates while lactoferrin, kallikrein, HSP-1 and an isoform of CRISP-3 were higher in seminal plasma of “bad freezers”.
Apart from the analysis of SP proteins, Barrier-Battut et al (2002) evaluated Ca, Mg, Cu and Zn concentrations in the SP of 15 stallions in relation to post-thaw quality of their ejaculates. No correlation between mineral levels and semen quality was observed even if the concentrations of some minerals were stallion related.
Although the studies presented in this paragraph are not conclusive, these results provide partial explanation on the high variety in susceptibility to cryopreservation between stallions. Further research is needed in order to understand whether it is possible or not to obtain reliable, repeatable and possibly predictive information on cryopreserved semen fertility from seminal plasma analysis.
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Evaluation of seminal plasma activities on the endometrium in vivo
While the positive effects of seminal plasma on sperm-PMN interactions were clearly demonstrated in vitro, the evidence that a modulation of PME also occurs in vivo is much harder to confirm. Some studies evaluated the influence of intrauterine infusion of SP alone or added to fresh spermatozoa on myometrial contractions at different time points after breeding. Portus et al (2005) found that mares bred in presence of seminal plasma had lower frequency of contractions and higher PMN number six hours after AI compared to mares bred with spermatozoa extended in skim milk. The proportion of these substances over the final volume of the AI dose was 87.5%. Whether the latter finding was a consequence of the first could not be determined, however the hypothesis that seminal plasma delayed uterine clearance could be refused in this study since mares presenting mild or significant fluid accumulation and pregnancy rates in the two groups did not differ (pregnancy rates were 76% for sperm-skim milk AI and 72% for sperm-seminal plasma AI). Pansegrau et al (2008) found that seminal plasma was not effective in improving myometrial contractions in subfertile mares bred twice, 24 and 48 hours after induction of ovulation. At the moment of AI mares received either oxytocin (20 UI, IV; OXY group) or intrauterine infusion of seminal plasma (62.5% on the final volume of the AI dose; SP group) or nothing (untreated group). The authors found that their score, based on frequency and strength of contractions, was similar and lower in SP and untreated groups than in OXY after the first AI, with OXY giving the same values of fertile mares used as control. After the second AI similar results were found with OXY showing significant higher score than SP. Moreover, on bacterial cultures performed 48 hours after the second AI, significantly more positive samples were obtained in SP compared to OXY thus suggesting that incomplete uterine clearance occurred in SP group. Since it was previously demonstrated that oxytocin is contained in all the fractions of the stallion's ejaculate but mainly in the gel (Watson et al, 1999) these findings can possibly be explained by the fact that gel is usually discarded by filtration and thus not included in the AI doses.
The effect of treatments that included SP on breeding induced endometritis in fertile mares were evaluated also on cytology, histology and on the expression of inflammatory cytokines. In a cross over design, Palm et al (2008) infused estrus mares with 5 ml of either PBS, seminal plasma, skim milk extender or egg yolk extender and checked the endometrium 12 hours later. Intrauterine fluid accumulation was detected in 3/32 cycles only and was not correlated
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with the treatment. Interestingly all the substances used, including PBS, caused endometritis according to both number of PMN and expression of inflammatory cytokines (TNF, IL-1b, IL-6) and COX-2, in comparison to untreated mares. No difference was observed between treatments. In a similar experiment Fiala et al (2002) evaluated endometrial cytology 4 and 24 hours after intrauterine infusion of 20 ml of either skim milk or seminal plasma. They found that while PMN number in the first treatment progressively increased until hour 24, when SP was infused this value peaked 4 hour later and then declined. This result might suggest that seminal plasma initially triggers PME, possibly in order to speed uterine clearance. The amount of SP used and the time points at which breeding induced endometritis was evaluated could have led to the different results in these latter two studies. However, pro or anti-inflammatory pathways triggered by seminal plasma could not be identified. Possibly the investigation of different cytokines could have led to other findings or perhaps the effects of seminal plasma on PME are more evident in presence of spermatozoa.
Fertility of cryopreserved semen after post-thaw addition of seminal plasma
Considering the positive effects that seminal plasma appeared to exert on both spermatozoa and the endometrium in vitro, some authors wondered whether post-thaw addition of SP immediately prior to AI would improve fertility. Alghamdi et al (2005) included five mares in a crossover design in order for them to receive both the following AI treatments in two separate cycles: frozen-thawed semen alone (1 billion spermatozoa, volume 2.5ml) and frozen-thawed semen plus 15ml of a homologous seminal plasma pool (85% SP on the final volume of the inseminate). Mares were inseminated at intervals of 12 hours starting 12 hours after hCG administration, thus on average 2.7 AI per cycle were performed. Pregnancy rates 15 days after ovulation were 20% (1/5) and 80% (4/5) for the two groups respectively. Panzani et al (2009) instead obtained 50% (4/8) and 42.9% (3/7) embryo recovery rate 8 days after ovulation in mares that were inseminated with frozen-thawed semen alone (250x106 spermatozoa, 2.5ml) or added of a homologous seminal plasma pool (2.5ml, 50% of the final volume of the inseminate). These two experimental designs present substantial differences concerning the number of inseminations per cycle, the timing of AI (24 hours after hCG administration in the second study), the insemination dose, the proportion of seminal plasma in which the sperm cells were resuspended post-thaw, the evaluation of fertility and the management of the mares after AI (Panzani et al performed a uterine lavage with 1 l of
