Post mating endometritis and pregnancy rate were not affected by the addition to frozen-thawed semen of filtered seminal plasma when mares without evidence of endometritis were artificially inseminated once, 40 hours post GnRH.
Chiara Sabatini, Alessandra Rota, Duccio Panzani, Matteo Tesi, Francesco Camillo
Veterinary Sciences Department, Pisa University, Via Livornese, 56124 San Piero a Grado, Pisa, Italy
Abstract
The main aim of this study was to evaluate the effect of the addition of seminal plasma to frozen-thawed semen on post mating endometritis and embryo recovery rate in mares when only one AI is done. Forty hours following induction of ovulation, 15 fertile Standardbred mares were submitted to a single artificial insemination (AI) per cycle with frozen semen obtained from one of two different stallions, on two cycles, according 2 different protocols: routine AI (200-280x106 frozen-thawed spermatozoa in 2 ml); and seminal plasma AI, (200-280x106 of frozen-thawed spermatozoa in 2 ml to which 7.8 ml of frozen-thawed seminal plasma was added). Six and 20 hours after AI, mares were evaluated by ultrasound for the presence of uterine fluid. Six hours after AI, mares were also subjected to uterine lavage for the evaluation of the presence and number of inflammatory cells. Eight days after ovulation, pregnancy was diagnosed by embryo recovery. There was a significant effect of treatment on subjective motility, which was lower when seminal plasma was added (20%; IQR: 10) compared to undiluted semen (45%; IQR: 10) (P<0.05). There was no significant effect of stallion or treatment on post mating endometritis or embryo recovery rate. In the mares and conditions of this study, the addition of seminal plasma to frozen-thawed semen had no effect on post AI uterine inflammation and pregnancy rate.
Keywords: mare; frozen semen; artificial insemination; seminal plasma; endometritis.
Corresponding author: Francesco Camillo, Department of Veterinary Sciences, Pisa University, Via Livornese, 56124 San Piero a Grado, Pisa, Italy. Tel +39 (0)50 2210162; email address: francesco.camillo@unipi.it
1. Introduction
cycle pregnancy rates (PR) resulting from AIs with frozen, cooled or fresh semen were reported to be 38-58% [2, 3], 39-87.5% [4, 5] and 55.1-84.6% [6, 7], respectively. Post-thaw semen quality is influenced both by the freezing process itself and by individual stallions differences. In fact, a great variation in semen quality and freezability exist between individuals, which in turn determine a great variation in fertility [8, 9].
Watson [10] 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 (SP) before cryopreservation. During the freezing process, spermatozoa undergo capacitation-like changes that can impair their ability to attach to oviductal epithelial cells in vitro [11]. A "decapacitating" action of seminal plasma (SP) was demonstrated in stallions [12] and rams [13]. Post-mating endometritis (PME) is a physiologic and temporary inflammation that develops as a response of the endometrium to the presence of spermatozoa [14]. Beside individual differences in susceptibility to PME, it has been clinically observed a more pronounced endometritis in mares submitted to AI with frozen-thawed semen [15]. Kotilainen et al [14] 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 fresh extended semen (5.0±4.4 x 106 PMN/ml). Since common procedures for semen cryopreservation include seminal plasma removal, the absence of this biological fluid could increase PME intensity and duration after breeding with frozen semen, thereby negatively affecting the chances to obtain a pregnancy.
On the other hand, stallion seminal plasma added post-thaw has shown several positive effects on spermatozoa, such as increased viability and acrosome membrane integrity [16]. Post-thaw semen characteristics of ‘bad- freezing’ stallions were improved by the addition of 5–30% of seminal plasma from stallions of proven fertility [17]. Seminal plasma components were also reported as modulators of the mating-induced endometritis by suppressing PMN binding and phagocytosis on viable sperm cells [18, 19]. Finally, the addition of seminal plasma to frozen thawed semen significantly improved fertility in swine [20] and tended to do so in mares [21] and jennies [22]. In the equine species, a beneficial effect was observed in vivo when seminal plasma was added to semen prior to an AI [21] done in mares which received a previous AI 4-12 hours earlier, thus with an already inflamed uterus. However, the increasing costs of frozen semen doses from valuable stallions makes today the use of more than one insemination dose per cycle almost impossible.
The main aim of this study was to evaluate the effect of the addition of seminal plasma to frozen-thawed semen on post mating endometritis and embryo recovery rate in mares when only one AI is done. The secondary aim was to evaluate if a spectrophotometer could be used instead of the Thoma counting chamber to evaluate the cellularity of uterine lavage fluids.
2. Material and methods
This study was performed at the Department of Veterinary Sciences, University of Pisa, from August to October.
2.1 Mares, estrous cycle and induction of ovulation
Fifteen Standarbred mares, aged between 5 and 11 years, in good body condition, and known to be fertile, were included. Mares were evaluated by ultrasound (US) on regular basis for oestrous cycle monitoring since the previous month of February without any clinical evidence of endometritis or uterine fluid accumulation. The mares were housed in paddocks and fed with hay ad libitum and a commercial horse fodder.
Ovarian activity was monitored daily by US during oestrus, beginning when a follicle of at least 30 mm was detected and up to ovulation. When the dominant follicle reached the diameter of 35 mm, in presence of uterine oedema and oestrus behaviour, ovulation was induced by a subcutaneous injection of 6 mg of the GnRH analogue buserelin (Suprefact®, Sanofi, Milano, Italia) and this time was considered hour 0 (h0, Table 1).
2.2 Stallions, frozen semen and seminal plasma
A total of five stallions of proven fertility after fresh and cooled semen AI, aged between 3 and 16 years, were employed: two provided semen (ST1F and ST2E) and three seminal plasma. The stallions were maintained in box and submitted to a balanced feeding.
Ejaculates were collected by a Colorado artificial vagina from stallions jumping on a phantom, submitted to routine evaluation and then either frozen as described by Vidament [23] or processed to extract seminal plasma. The sperm concentration prior to freezing was 100-140x106 spermatozoa/ml, corresponding to 50-70x106 spermatozoa in each 0.5 ml straw. To obtain seminal plasma, ejaculates were centrifuged at 700 g for 10 min and the supernatant was passed through a
series of sterile filters from 5.0 to 0.22 μm (Sartorius Minisart, Aubergne, France) and stored at -18°C until use.
2.3 Artificial Insemination
Mares were inseminated 40 hours after the induction of ovulation (Table 1) according to two different treatments: routine AI (RAI) or AI with addition of seminal plasma to the thawed sperm cells (SPAI).
Immediately before thawing the straws, for SPAI treatment, 2.6 ml of SP from each of the 3 stallions were thawed in a water bath at 37°C and pooled in a sterile tube kept at the same temperature.
Four straws of frozen semen of the assigned stallion were thawed in water bath at 37°C for 30’’, dried and opened in a empty (RAI treatment) or containing SP (SPAI treatment) sterile tube kept at 37°C. Semen was evaluated for subjective total motility under a phase contrast microscope at 37°C, by a single operator blinded to treatment, before being released in the body of the uterus of the designed mares.
Insemination dose was 200-280x106 spermatozoa in both treatments, while inseminated volume was 2 ml in RAI and 9.8 ml in SPAI, respectively.
Each mare was bred with semen from a single stallion on two cycles, one for each of the two treatments. Stallions and order of treatment were randomly assigned.
Mares that ovulated earlier than 36h after induction of ovulation were not inseminated, and mares that ovulated later than 60 hours after induction of ovulation were excluded from the results.
2.4 Evaluation of post mating endometritis and uterine cytology
Six hours after AI (h 46, Table 1), mares were checked for presence and degree of PME by both ultrasound and uterine lavage. The amount of fluid observed on US was scored as ≤ or > 2 cm, while its echogenicity was scored from 4 (anechoic) to 1 (very echoic) [24]. US evaluation, but not uterine lavage, was repeated at h 60 (corresponding to 20 hours after AI, Table 1).
The uterine lavage was performed infusing and recovering in the same bag 1 L of Ringer Lactate (Galenica Senese, Siena, Italy) pre-warmed at 37°C. The bag was weighed in order to assess the volume of fluid recovered. Then, after mixing, 10 ml of lavage fluid were aspirated with a syringe and subjected to the analysis of the adsorbance and to the total cell count. Adsorbance was determined by a spectrophotometer (Accucel, IMV-Technologies Italy, Piacenza, Italy). First, 2 ml of
Ringer Lactate were used to calibrate the machine, then 2 ml of lavage fluid were evaluated twice for adsorbance. Mean value was recorded. Total cells concentration was assessed by direct count on a Thoma counting chamber and recorded as number of cells per ml.
Meanwhile the bag was hanged up for 30 minutes to allow cells to precipitate. After precipitation, 20 ml of the settled fluid were collected from the bottom of the Ringer bag. After gentle mixing a 200 µl drop was placed on a glass slide and allowed to dry (Smear 1).
The remaining fluid was centrifuged at 600 g for 10 minutes, the supernatant was discarded and 200 µl of the pellet were used to prepare a second smear as described above (Smear 2).
Both smears were stained by Diff Quik. On Smear 1, the percentage of neutrophils was determined by a single operator blinded to treatment by direct observation of 200 cells under a bright field microscope at 1000x magnification. Consequently, the number of neutrophils/ml in the overall recovered fluid was obtained by the following proportion: % neutrophils : 100 = x : total cell count (Thoma). On Smear 2, the presence of eosinophils was estimated by recording the number of these cells in 50 fields.
2.5 Embryo recovery rate
Eight days after ovulation, embryos were recovered by uterine flushing as previously described [25] (Table 1). Mares were then treated with a PGF2alpha analogue (Alfaprostol®, 3mg IM, Gabbrostim, Ceva Italy, Agrate Brianza, Italy) in order to induce a new oestrus.
8 PM 8 AM 12 AM 6 PM
Day 0 US+GnRH, h0
Day 1 US, h12
Day 2 US, h36 US+AI, h40 US+Lavage, h46
Day 3 US, h60
Day 11 Embryo recovery
Table 1: Timeline of the study.
2.6 Statistical analysis
Normal distribution of data was assessed by the Anderson-Darling test. Since data did not followed a normal distribution, a two tailed Wilcoxon Signed Rank Test was used to determine the effects of treatment and stallion on the evaluated parameters. A linear regression was used to evaluate the relation between adsorbance and cell concentration. Differences in presence of intrauterine fluid
after AI and embryo recovery rate between the two treatment groups were analysed by the Chi Square test. Data are presented both as median and interquartile range (IQR) and as mean and standard deviation. Differences in embryo recovery rates were evaluated by Fisher’s exact test. Level of significance was set at P<0.05.
3. Results
3.1 Semen quality
There was a significant effect of treatment on subjective motility, which was lower when semen was added of seminal plasma (20%; IQR: 10; SPAI) than in undiluted semen (45%; IQR: 10, RAI) (P<0.05). Average motility within treatments did not differ between stallions (P>0,05) being equal to 16% (IQR 10) and 46% (IQR 10) for ST1F and to 26% (IQR 15) and 49% (IQR 10) for ST2E in SPAI and RAI respectively.
3.2 Evaluation of post mating endometritis and uterine cytology
Presence of uterine fluid (<2 cm) 6 hours after AI was observed in 2/15 mares for RAI (echogenicity 4-3) and in 4/15 mares for SPAI (echogenicity 4-2). Twenty hours after AI, corresponding to 14 hours after uterine lavage, uterine fluid was still present in 3/15 (echogenicity 4-2) and 6/15 (echogenicity 4-3) mares for RAI and SPAI, respectively. Neither the echogenicity nor the amount of fluid differed significantly between treatments within the two time intervals or overall, however 6 hours after AI intrauterine fluid was detected more often in mares that were inseminated with ST1F than in mares inseminated with ST2E (P<0.05). This effect was no longer observed 20 hours after AI.
Data related to uterine lavage and cytology within each treatment are shown in Table 2 Differences between treatments and between stallions were not statistically significant (P>0.05). When evaluating the cellularity of the uterine lavage fluid, a significant relation was found between the values obtained by the spectrophotometer and the Thoma counting chamber, expressed by the following regression equation Y = 4.272*X - 0.3324 (P< 0.0001) (Figure 1).
Routine AI Seminal plasma AI Median (IQR) mean Median (IQR) mean
(range) ±SD (range) ±SD Weight of the Ringer bag (g) 880 (160)
(350-910) 810 ±156 840 (210) (210-920) 757 ±208 Adsorbance 0.08 (0.44) (0.04-1.17) 0.30 ±0.37 0.29 (0.22) (0.04-1.05) 0.32 ±0.24 Cells/ml (x106) 0.20 (1.29) (0.01-6.38) 1.00 ±1.73 0.77 (0.73) (0.007-4.32) 1.00 ±1.05 % PMN 94 (28.5) (25-98) 79.17 ±24.57 97 (3.5) (10-99) 89.11 ±23.35 PMN/ml (x106) 0.19 (1.26) (0-6.19) 0.95 ±1.67 0.75 (0.83) (0-4.23) 0.93 ±1.05 EOS/50f 2 (47) (0-225) 35.20 ±67.02 10 (11) (0-50) 15.07 ±16.36 Table 2: Uterine lavage and cytology parameters evaluated after routine frozen semen AI (n=15) and AI with frozen semen added of seminal plasma (n=14). Data are presented both as median, interquartile range and as mean and standard deviation (P>0.05). PMN: polymorphonuclear granulocytes; EOS: eosinophilic granulocytes
Fig 1: Linear regression between absorbance and cell concentration evaluated by hemocytometer
3.3 Time of ovulation and embryo recovery rate
Six mares ovulated before hour 36 from induction of ovulation and weren’t inseminated. Five mares did not ovulate after AI: four presented a persistent anovulatory follicle [26] and one an atresic follicle. These mares were excluded from the analysis of the fertility results. The distribution of first ovulations, in the cycles included in the analysis of fertility, was as follows: 11/30 mares ovulated between 36 and 40 hours after GnRH treatment, 18/30 between 40 and 46 hours and 1/30 between 46 and 60 hours (Table 3).
DAI Seminal plasma ST1F 1 NO
DAI Routine ST1F 1 YES
ESE Seminal plasma ST1F 1 YES
ESE Routine ST1F 1 NO
ELD Seminal plasma ST1F 1 NO
ELD Routine ST1F 1 NO
IOL Seminal plasma ST1F 3 YES
IOL Routine ST1F 1 NO
MAR Seminal plasma ST1F 1 YES
MAR Routine ST1F 1 NO
FRO Seminal plasma ST1F 1 NO
FRO Routine ST1F 1 NO
GHI Seminal plasma ST1F 1 NO
GHI Routine ST1F 1 NO
ITA Seminal plasma ST2E 1 NO
ITA Routine ST2E 2 YES
GOR Seminal plasma ST2E 2 YES
GOR Routine ST2E 2 NO
FIF Seminal plasma ST2E 1 YES
FIF Routine ST2E 2 NO
LAM Seminal plasma ST2E 1 YES
LAM Routine ST2E 1 YES
GIO Seminal plasma ST2E 1 NO
GIO Routine ST2E 1 YES
IMP Seminal plasma ST2E 2 NO
IMP Routine ST2E 1 1 YES
DOU Seminal plasma ST2E 1 1 NO
DOU Routine ST2E 1 NO
LOI Seminal plasma ST2E 1 NO
LOI Routine ST2E 1 1 YES
Table 3: Time of ovulation, stallion employed and occurrence of embryo recovery after routine frozen semen AI or AI with frozen semen added of seminal plasma in mares. AI was done at hour 40 after GnRH injection.
ov36-40 = ovulation occurred between 36 and 40 hours after GnRH injection ov40-46 = ovulation occurred between 40 and 46 hours after GnRH injection ov46-60 = ovulation occurred between 46 and 60 hours after GnRH injection
Embryo recovery rates per cycle and per ovulation were the same for the two treatments (Table 4). Similarly, no differences were observed between stallions (Table 5), however the embryo recovery rate tended to be higher for one stallion when only the RAI treatment was considered (P=0.057). Overall, there was no statistical difference between the embryo recovery rate when insemination occurred 0-4 hours post ovulation or 0-6 hours pre ovulation (Table 6).
Routine AI (%) Seminal plasma AI (%)
Embryos/cycles 6/15 (40) 6/15 (40)
Embryos/ovulation 6/20 (30) 6/20 (30)
Table 4: Embryo recovery rates after routine frozen semen AI and AI with frozen semen added of seminal plasma in mares.
Routine AI (%) Seminal plasma AI (%)
ST1F ST2E ST1F ST2E
Embryos/cycles 1/7 (14) 5/8 (62) 3/7 (43) 3/8 (37)
Embryos/ovulation 1/7 (14) 5/13 (38) 3/9 (33) 3/11 (27)
Table 5: Embryo recovery rates for the two stallions after routine frozen semen AI and AI with frozen semen added of seminal plasma in mares.
Type of AI ov36-40 (%) ov40-46 (%)
Embryos/cycles Routine AI 2/4 (50) 4/10 (40)
Seminal plasma AI 2/7 (29) 4/8 (50)
Total embryos/cycles Routine+ Seminal plasma AI 4/11 (36) 8/18 (44) Table 6: Time of ovulation and occurrence of embryo recovery after routine frozen semen AI or AI with frozen semen added of seminal plasma in mares. AI was done at hour 40 after GnRH injection. One mare, not included in this table, ovulated between 46 and 60 hours after GnRH.
ov36-40 = ovulation occurred between 36 and 40 hours after GnRH injection ov40-46 = ovulation occurred between 40 and 46 hours after GnRH injection
4. Discussion
Equine seminal plasma has been widely studied with respect of its composition and of its activities on both the spermatozoa and the endometrium [16, 17, 18, 19, 21]. Beneficial effects in terms of sperm function and modulation of post mating endometritis were reported in the literature, as discussed later. Since seminal plasma has to be removed in order to make semen freezing possible [27] its post-thaw restoration to the semen dose may improve the outcome of frozen semen artificial inseminations. Unfortunately, despite the considerable amount of “in vitro” findings, scarce are the studies to date made to evaluate whether seminal plasma can affect pregnancy or embryo recovery rate in the horse or not. Alghamdi et al. [21] found that post-thaw restoration of seminal plasma (85% of the volume of the AI dose) improved pregnancy rates of mares bred with frozen semen (4/5 pregnancies compared to 1/5 when seminal plasma was replaced by a commercial extender). Panzani et al. [28] found no difference comparing embryo recovery rates following AI with post-thaw addition of 50% SP and conventional AI: three/7 and 4/8
embryos/cycles were recovered, respectively. Similarly, post-thaw addition of 80% SP in the present study did not improve embryo recovery rate neither per cycle nor per ovulation, compared to conventional AI. However, while per cycle embryo recovery rate in RAI treatment tended to be higher for the stallion ST2E (5/8 embryos) compared to ST1F (1/7, P=0.057), this trend was no longer observed in SPAI treatment. This difference might have been confirmed if a higher number of AIs would have been performed.
Fertility results obtained so far in other animal species were overall more encouraging and appeared to be related to the SP proportion used. In sheep, no effect of post-thaw semen resuspension in 30% seminal plasma was seen [29] while a concentration of 50% significantly increased litter size when ewes were inseminated at the peak of the breeding season, although the number of lambing ewes was not significantly higher [30]. In pigs, when frozen-thawed spermatozoa from low fertility boars were resuspended in 10% seminal plasma from fertile boars, conception rate was significantly improved [20] and, in another study, the reduction of litter size observed in sows inseminated with frozen compared to fresh semen was not observed when 50% seminal plasma was added post-thaw [31]. One study in the dog showed that fertilization rate and litter size increased when bitches were inseminated with frozen semen in presence of approximately 85% seminal plasma [32].
Similar studies were also performed in donkeys, a more closely related species to the horse, for which the very low pregnancy rates obtained with frozen semen AI (0-30%) is a generally recognized problem and represents an important obstacle toward the preservation of the endangered breeds of this species by sperm banking. In donkeys, when semen frozen with glycerol was diluted post-thaw with homologous seminal plasma (70% of the AI dose volume), pregnancy rate increased to around 60% [22]. This result, although not statistically higher than what found for conventional AI or AI with post-thaw extender restoration [22], is the highest fertility outcome obtained so far with donkey cryopreserved semen.
It has been demonstrated in vitro that SP reduces sperm binding to polymorphonuclear cells (PMN) [33] and that some of its components (proteins such as CRISP3 and lactoferrin) are able to direct PMN phagocytosis against dead spermatozoa only, thereby protecting live sperm cells during post mating endometritis [34, 19, 35]. These findings suggest that seminal plasma might modulate PME, however this hypothesis has still to be confirmed in vivo. Unfortunately, the only study in
which frozen semen fertility was improved by seminal plasma [21] does not provide data on PME. In the present study, similarly to Panzani et al. [28], uterine fluid accumulation was similar in mares inseminated with frozen semen alone or frozen semen added of seminal plasma.
Uterine lavage is commonly used for the evaluation of endometrial cytology: a sample representative of the whole uterine surface is recovered [36] and can be further processed for qualitative and/or quantitative analysis such as cell count and identification. The hemocytometer, although is the gold standard to evaluate cell concentration, requires direct visualization by the operator to count the cells. According to the results of the present study, the spectrophotometer used to evaluate semen concentration, can be employed to evaluate cell concentration in uterine lavage fluid. This instrument thus offers a less time consuming and a not operator-dependent analysis, even though it might lack of accuracy in case of strong contamination of the sample (e.g. extracellular debris, mucus, blood).
In this study, no difference was found between SPAI and RAI treatments in PMNs present in the uterine fluid. Portus et al. [37] inseminated mares with fresh semen added of either seminal plasma or extender (87.5% for both) and observed 6 hours later that the first group resulted in a higher PMN number together with decreased uterine contractions. Fiala et al. [38] monitored PME at 4 and 24 hours after intrauterine infusion of either seminal plasma or skim milk extender alone, without spermatozoa, and interestingly found that in the SP treatment PMN number peaked at hour 4 and then declined, while in the skim milk treatment it progressively increased until hour 24 [38]. In donkeys, post-thaw addition of seminal plasma, compared to an extender, has not increased PMN number 6 hours after AI in one study [39] while Rota et al. [22] found a higher PMN concentration both 6 and 10 hours after AI. When only one uterine lavage was performed 24 hours after AI this difference was not observed [40].
In conclusion, taking these results all together, it is difficult to understand if post-thaw restoration of seminal plasma prior to AI may be beneficial in the horse. Although the present study failed to demonstrate that seminal plasma decreases the incidence of PME and increases pregnancy rates, it may suggest that there might be stallion-dependent differences.
Acknowledgements
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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