Sperm Production and Sperm Morphology of Swedish Warmblood Stallions

The present study investigated daily sperm output and sperm morphology of fresh semen in eight Swedish Warmblood stallions aged 5–8 years. They were used for artificial insemination, and their fertility during the breeding season of semen collection exceeded 60% per cycle. One ejaculate of semen was collected daily for 10 consecutive days from each stallion. The gel-free volume was measured, and the sperm concentration was assessed with a Bürker chamber. The volume of gel-free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). Sperm morphology was examined in ejaculates collected on days 2, 5 and 10. An aliquot from each ejaculate was fixed in 1 ml formol–saline immediately after collection and examined under a phase-contrast microscope (magnification 1000×) to assess morphological abnormalities. Furthermore smears were prepared and stained according to Williams (carbolfuchsin–eosin) for a more detailed examination of the sperm heads under a light microscope (magnification 1000×). Analysis of variance was applied to data. Total spermatozoa number decreased progressively during the first 8 days of collection, and daily sperm output (DSO) was calculated as mean TSN of collections on days 8–10, being 6.4 × 10⁹ spermatozoa. The overall percentages of morphologically normal spermatozoa in ejaculates collected on days 2, 5 and 10 were above 70%, being significantly lower in ejaculate 2 (68.6%) compared with ejaculates 5 and 10 (72.9% respectively 75.3%).     

Testicular Measurements and Daily Sperm Output of Tori and Estonian Breed Stallions

Evaluation of testicular measurements and daily sperm output (DSO) yields valuable information for predicting the reproductive capacity of stallions. The present study evaluated testicular measurements (height, length, width and circumference) and DSO of eight Tori and eight Estonian breed stallions. One ejaculate of semen was collected daily for 10 subsequent days from each stallion. The gel‐free volume of semen was measured with a graduated glass cylinder and the sperm concentration was assessed with a Chorjajev chamber. The volume of gel‐free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). The DSO was calculated as mean TSN of collection on days 8–10 in Tori breed stallions and on days 4–10 in Estonian breed stallions. The DSO of Tori breed stallions was 12.9 × 10⁹ spermatozoa and of Estonian breed stallions 4.5 × 10⁹ spermatozoa (p < 0.001). Testicular measurements (in cm) 1 day after the last semen collection were as follows: left testis– height 7.3, length 10.4 and width 7.3 in Tori breed stallions, and 5.9, 8.1 and 5.9, respectively, in Estonian breed stallions; right testis– height 7.4, length 10.6 and width 7.4 in Tori breed stallions, and 5.5, 7.4 and 5.3, respectively, in Estonian breed stallions. All these testicular measurements were significantly smaller in Estonian than in Tori breed stallions (p < 0.001). Testicular circumference was 45.4 and 35.4 cm in Tori and Estonian breed stallions, respectively (p < 0.001). The testicular circumference was correlated with DSO in both Estonian (p < 0.05) and Tori breed stallions (p = 0.071). The results give us valuable information on the reproductive capacity of Tori and Estonian breed stallions.     

Level of Glycolyzable Substrates in Stallion Semen: Effect of Ejaculation Frequency on Sperm Survival after Cool Storage during the Nonbreeding Season

Although seminal characteristics are routinely evaluated in the stallion, the effect of collection schedules and seminal plasma on semen quality during cool storage is not well understood, specifically during the nonbreeding season when cryopreservation of stallion semen is preferentially performed. To address these issues, behavioral characteristics, seminal parameters, and biochemical markers (d-glucose, fructose, and citric acid) were measured in ejaculates (n = 60) obtained during the nonbreeding season. Semen was collected from three stallions, twice a day (1-hour gap between successive collections) and two times in a week. Differences between the means of first and second ejaculates were observed for erection latency (P < .001), which was higher in second ejaculates and determined a higher total breeding time (P < .1). Variations introduced by the stallion were significant for number of mounts (P < .05, in first ejaculates), erection latency (P < .001, in second ejaculates), and total breeding time (P < .001, in second ejaculates). First and second ejaculates differed significantly for sperm motility and sperm concentration (P < .001, higher in first ejaculates) and pH (P < .01, higher in second ejaculates). d-glucose was present in seminal plasma at a much higher concentration than fructose (P < .001) in both ejaculates. There were no significant stallion-associated differences in sperm vitality and pH in the first and second ejaculates as well as in sperm concentration for the second ejaculates. The effect of seminal plasma on equine sperm survival during cooled storage was analyzed by monitoring sperm motility and cell morphology after conservation in an extender medium with and without seminal plasma. When statistically considering seminal plasma and conservation time simultaneously, it was found that these variables affected acrosomal status and midpiece morphology.     

Phenotypic Correlations of Testes Size with Semen Traits and the Productive Traits of Young Boars

The aim of this study was to investigate whether there is a relationship of young boar testes size with semen traits and with productive traits. The dimensions (length, width and volume) of each testis and semen traits (semen volume, percentage of sperm with progressive motility, sperm concentration, total number of sperm in semen, percentage of sperm with normal acrosome, percentage of sperm with major and minor morphological defects, osmotic resistance test value and activity of aspartate aminotransferase in seminal plasma) were determined on 120 young boars aged 6 months. At 180 day of age, the boars backfat thickness and leanness (by ultrasonic apparatus) and body weight were also measured. The average daily gain was determined in the period from 70 to 180 days of age of the boars. The results showed that the sperm concentration, total number of sperm in semen and percentage of progressive motile sperm were a significantly positively correlated with width and volume of the left (p ≤ 0.01) and right testis (p ≤ 0.05) and with total volume of both testes (p ≤ 0.01). But the highest values of correlation coefficients were found between the width of the left testis and sperm concentration, total number of sperm in semen and percentage of progressive motile sperm. A correlations of dimensions (length and width) and volume of testes with other semen traits were very low and statistically non‐significant. The volume of testes (left and right testis and total testes) was significantly positively correlated with body weight at 180 days of age and daily gain (p ≤ 0.01), but lower correlation coefficient was between left testis and daily gain (p ≤ 0.05), whereas correlations were low and non‐significant with leanness and backfat thickness.     

Hemicastration and castration in the beagle dog; the effects on libido, peripheral plasma testosterone concentrations, seminal characteristics and testicular function

Hemicastration of five beagle dogs had no effect on their libido, and in four dogs the total seminal volume and sperm concentration of the second fraction was unchanged; sperm output immediately fell in these four animals. Plasma testosterone concentrations in the peripheral circulation were largely unaffected by hemicastration but following complete castration the value fell rapidly and remained low. The first ejaculates, collected 5 days after complete castration, were aspermic. There was no evidence of compensatory changes in the histological structure in those testes which were removed 41 to 44 days after the first gonad. Evidence of the maturation of spermatozoa with migration and subsequent detachment of the cytoplasmic droplet was observed in stained smears of epididymal semen.     

Seminal characteristics of two- and three-year-old quarter horse stallions

Seminal and testicular characteristics were compared in 19 three-year-old and 23 two-year-old Quarter Horse stallions. Semen was collected, and testicular evaluations made on all stallions once in April or May and again 60 days later. Semen was collected from stallions twice, one hour apart, at each evaluation. Average testicular tone and scrotal width were greater in three-year-olds than in two-year-olds. Ejaculates of three-year-olds had higher motility scores (66% vs 47%) sperm movement (3.2 vs 2.4), normality (77 vs 71), and total number of normal, motile spermatozoa per ejaculate (4.3 × 10⁹ vs 2.2 × 10⁹). Two-year-olds had twice as many cytoplasmic droplets (11% vs 5%) as three-year-olds. Average sperm concentrations per ml of gel-free semen and gel-free volume of ejaculates were not different. Volume of ejaculate and motility of spermatozoa were positively correlated with pregnancy rate, whereas percent cytoplasmic droplets was negatively correlated with pregnancy rate in both groups of stallions. Pre- and post-ejaculatory urethral, seminal, and preputial cultures were examined for pathologic organisms on all stallions. Eighteen of 42 had positive cultures for Klebsiella spp., Pseudomonas spp., β-Strep spp., or E. coli. Nine two-year-olds and 3 three-year-olds had positive cultures for Klebsiella spp. All but one of these stallions were housed on wood shavings and allowed limited exercise. Three-year-old stallions had superior seminal quality as compared with two-year-olds.     

Semen parameters and hormone concentrations in stallions subjected to long-term estrogen administration

Eight pony stallions were paired by estimated daily sperm output (DSO) and randomly assigned to one of two treatments in a randomized block experiment. Stallions received 44 μg/kg BW estradiol cypionate (ECP) or an equivalent volume of physiological saline solution on alternate days during the breeding season. Blood samples collected immediately preceding each injection were assayed for luteinizing hormone (LH), estradiol-17β (E₂) and testosterone (T). Semen was collected twice weekly, 3.5 days apart, to evaluate sperm motility and total number of sperm per ejaculate. Prior to and after 4, 8 and 12 weeks on treatment, semen was collected once daily for 7 days to determine DSO. Data were separated into 9 periods (10 days each) for statistical analysis and subjected to analysis of variance for a randomized block design to determine treatment effects.There were no differences (p>.05) between groups for DSO or LH prior to initiation of treatment. Testosterone was higher (p<.05) in ECP stallions compared with C stallions prior to treatment and at all time points measured. As expected, E₂ was higher (p<.05) in the ECP stallions compared to C stallions after 20 days (period 2) of treatment and for the remainder of the experiment. However, E₂ was higher (p<.05) in the C group prior to treatment, but there was no difference between the groups after 10 d of treatment (period 1). ECP stallions had higher (p<.05) DSO than C stallions after 30 d on treatment. After 40 and 50 d (periods 4 and 5), ECP stallions demonstrated higher (p<.05) total sperm per period than C stallions. This was preceded by higher (p<.05) LH values for ECP stallions than for C stallions after 10 and 20 d (periods 1 and 2). No differences were found between the ECP and C groups for LH between 30 and 60 d. Although numerically higher, no significant differences (p>.05) were seen after 60 days for DSO or after 60, 70 or 80 days for total sperm per period. ECP stallions had higher (p<.05) DSO and total sperm per period after 90 d than C stallions. Additionally, LH remained significantly higher (p<.05) in the ECP group after 60 days (periods 7, 8 and 9). Elevated LH concentrations in ECP stallions demonstrated that estrogen treatment did not inhibit LH secretion in this study.     

Semen quality and fertility after heat stress in boars

Sperm morphology and the fertilizing capacity of ejaculated spermatozoa were examined in 6 Swedish Landrace boars before and after heat stress. The boars were exposed to 35° C during 100 h in a climatic room. Fertility was measured by insemination of gilts before and at various times after heat stress. Each gilt (n = 44) was inseminated with a total of 5×10⁹ spermatozoa diluted to 10O ml with EDTA-glucose diluent and fertilization was assessed by examining recovered ova 2 days after insemination.Changes in semen quality varied among the boars from a very weak response in 2 boars to pronounced semen alterations occurring 2–6 weeks after heat stress in the other boars. A close relationship was found between seminal changes and fertilization rates, all ejaculates which had high fertilization rates being of the same quality as the pre-exposure ejaculates. The ejaculates that had poor fertility were characterized by lowered sperm motility and increased numbers of spermatozoa with abnormal heads, proximal cytoplasmic droplets and nuclear pouch formations.     

The Relationship between Semen Quality and the Nonreturn Rate of Bulls

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The value of routine evaluation of bull semen was analysed for 117 AI bulls placed in two studs. Data from semen analysis of a total of 1635 ejaculates was compared statistically with the nonreturn rates of the bulls. The semen parameters which were significantly correlated to nonreturn rates were the motility of the freshly collected ejaculates (p = 0.0140) and post-thaw motility (p = 0.0075). The total number of motile sperm in the inseminate ranged from 10.9 to 19.3 × 10⁶ and according to previous reports the effects of low motility should be fully 'compensated' when doses above 10 × 10⁶ sperm/dose are used for insemination. In conclusion, the motility of freshly collected semen does not appear to be 'compensation' and a low percentage of motile sperm in an ejaculate may indicate other dysfunctions of the population of motile cells. Furthermore, post-thaw motility appears to correlate significantly with nonreturn rates. The largest proportion of the variation was explained by the breed of the bull and stud (42.2% of the variation), whereas the two motility parameters explained 10% of the total variation in nonreturn rates. Objective and precise evaluation of sperm motility in combination with other semen traits are needed to improve breeding efficiency. Although microscopic evaluation of sperm motility correlates with nonreturn rates of bulls, the methods are subjectively assessed and inaccurate and therefore do not allow a satisfactory prediction of fertility.     

Clinical observations on changes in concentrations of hormones in plasma of two stallions with thermally-induced testicular degeneration

To investigate effects of thermally-induced testicular degeneration on hormonal and seminal parameters in stallions, the scrotum was insulated for 36 hours in two mature (5-year-old mixed breed and 11-year-old Throughbred) stallions. Semen was collected daily for 10 days (DSO) prior to, and at intervals after, scrotal insulation. When DSO determinations were not being made, semen was collected 3 times weekly. Jugular blood samples were collected at 15-minute intervals for 6 hours from each stallion prior to, and at intervals after, scrotal insulation. A mouse interstitial cell testosterone assay was modified to quantify biologic activity of equine luteinizing hormone (BLH) in plasma samples. Immunoactive luteinizing hormone (ILH) and testosterone (T) concentrations were determined in plasma samples by routine RIA procedures. Percentages of progressively motile and morphologically normal spermatozoa began to decrease by 1 to 2 weeks postinsulation, reached nadir values at 3 to 3-1/2 weeks postinsulation, and returned to preinsulation values by 7 weeks postinsulation. Total number of spermatozoa and total number of progressively motile, morphologically normal spermatozoa in ejaculates at DSO returned to normal by 8 weeks postinsulation in stallion 2 and 12 weeks postinsulation in stallion 1. Concentrations of BLH and ILH increased, and while T concentrations decreased, immediately postinsulation. The increase in ILH concentrations was greater than the increase in BLH concentrations, resulting in a decrease in the BLH:ILH (B:I) ratio. Following the peak in LH secretion immediately postinsulation, LH concentrations gradually decreased while T concentrations increased. The B:I ratio was elevated from 1 to 13 weeks postinsulation compared to immediately postinsulation. In addition to changes in spermatozoal quality in ejaculates, stallion response to scrotal insulation included increased secretion of luteinizing hormone and impaired Leydig cell function (as determined by reduced testosterone concentration in circulating plasma). The proportion of biologically active LH secreted in response to thermal testicular injury increased during the recovery phase.     

Factors influencing semen characteristics in boars

Factors influencing semen characteristics in young boars reared in a subtropical environment were studied. Age had only a slight effect on semen volume, but had a significant effect on sperm concentration, total sperm in the ejaculate and daily sperm output (DSO), there being marked increase in boars over 12 months of age compared with younger boars. Boars aged between 8 and 12 months had a DSO of 8.1 +/- 2.6 X 10(9) compared with boars 13 to 15.5 months with a DSO of 14.3 +/- 3.9 X 10(9) and boars aged 16 to 18 months with a DSO of 15.2 +/- 6.2 X 10(9). A highly significant correlation was found between bodyweight and all the semen characteristics examined, the highest correlation being with total number of sperm in the ejaculate. Daily sperm output was not significantly correlated with testicular size as measured by width or length in the live animal. Copulatory behavior had little influence on semen characteristics. Semen characteristics studied were not found to be adversely affected by season. A highly significant correlation between total volume and fluid volume, total volume and gel volume, and total volume and sperm concentration was found, but no correlation was found between total volume and total sperm in the ejaculate. Fluid volume was correlated significantly with gel volume, sperm concentration and total sperm in the ejaculate. A highly significant correlation was found between sperm concentration and total sperm in the ejaculate.     

Numbers of Sertoli cells, quantitative rates of sperm production, and the efficiency of spermatogenesis in relation to the daily sperm output and seminal quality of young beef bulls

Data from 34 yearling Hereford or Angus bulls were used to investigate relationships of testicular size, quantitative rates of sperm production, Sertoli cell numbers, numbers of germ cells supported per Sertoli cell, and the efficiency of spermatogenesis to daily sperm output and seminal quality. Two ejaculates were collected by electroejaculation from each bull on each of 2 days/week throughout the study. The percentage of progressively motile sperm and the percentage of morphologically normal sperm were determined from aliquots of fresh semen. Additional aliquots of semen were frozen in glass ampules or plastic straws and subsequently evaluated for postthaw motility and percentage of sperm with intact acrosomes. Sertoli cell numbers, the numbers of germ-cells per Sertoli cell, and the efficiency of spermatogenesis were unrelated to the quality of fresh or frozen semen (P greater than 0.05). In first ejaculates, the numbers of sperm and motile sperm were related (P less than 0.05) to testicular parenchymal weight (r = 0.38 and 0.50), daily sperm production (r = 0.45 and 0.53), and spermatids per gram of testicular parenchyma (r = 0.35 and 0.34). Testicular parenchymal weight and daily sperm production also were related to daily sperm output and to the average daily motile sperm output of these bulls (P less than 0.05), but could account for less than 25% of the variability in these end points among bulls.     

Cholesterol Loaded Cyclodextrin Supplementation Enhances the Cholesterol-to-Phospholipid Ratio and Diminishes Oxidative Stress in Jack Spermatozoa During Cryopreservation

The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5, 2, and 3 mg/mL CLC to obtain 120 × 10⁶ sperm/mL spermatozoa concentration. The semen was cryopreserved using customized freezing protocols. Evaluation of seminal parameters, the C:P ratio, and the oxidative status of jack spermatozoa was analyzed at all stages of cryopreservation. The oxidative status in the jack semen was evaluated by measuring malondialdehyde, glutathione and total antioxidant capacity levels. The results indicated that the mean percent values for various seminal quality parameters and the oxidative parameters were found to be significantly higher (P < .05) in CLC-treated groups with the highest values for 2 mg of CLC/120 × 10⁶ spermatozoa. In conclusion, the present study revealed that the supplementation of CLC before cryopreservation has significantly reduced the oxidative stress and also increased the C:P ratio during semen cryopreservation process. Furthermore, a reduction in lipid peroxidation levels, reduced damage to the sperm plasma and acrosome membranes and improvement in the post-thaw sperm integrity as well as stability were recorded.     

Alkaline and Acid Phosphatase Activity, pH and Osmotic Pressure of Boar Semen

Alkaline phosphatase activity was recorded in forty ejaculates of the sperm rich fraction of boar semen as 9,790 ± 5,250 Klein-Babson-Read units per 100 ml. of seminal plasma. Acid phosphatase activity in the same ejaculates was 681 ± 304 Babson-Read units per 100 ml. of seminal plasma. No alkaline phosphatase activity was detected in the seminal plasma of vasectomized boars.

The pH of the sperm rich fractions was 7.69 ± 0.33 and the osmotic pressure was 313.56 ± 7.98 milliosmols.

     

Electroejaculation Increases Low Molecular Weight Proteins in Seminal Plasma Modifying Sperm Quality in Corriedale Rams

This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and 2‐D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation.     

Reproductive Development of Santa Inês Rams During the First Year of Life: Body and Testis Growth,Testosterone Concentrations,Sperm Parameters,Age at Puberty and Seminal Plasma Proteins

We have investigated the reproductive development of the tropically adapted Santa Inês ram, the most common hair sheep in Brazil. From 8 to 48 weeks of age, 16 animals were evaluated for body and testis growth, semen parameters, testosterone concentrations and seminal plasma proteins, using two‐dimensional SDS‐PAGE. Animals were weaned at 30 days and kept in feedlots thereafter, receiving hay, concentrate (18% of crude protein) and mineral supplement. Body weight increased from 12.3 ± 0.7 to 54.3 ± 1.6 kg between 8 and 48 weeks (p < 0.05), but changes in thoracic perimeter and scrotal circumference were non‐significant after 36 weeks (p > 0.05). The percentage of motile sperm increased slowly until 23 weeks and more rapidly after that age, but significant changes in progressive motility occurred after 25 weeks. Presence of abnormal sperm related inversely to age. Most significant changes in sperm concentration occurred between 38 and 44 weeks (0.38 ± 0.05 to 1.14 ± 0.24 × 10⁹ cells/ml, p < 0.05) and testosterone reached its highest concentrations at 42 weeks, decreasing afterwards. Rams reached puberty at 28.2 ± 0.8 weeks. The number of protein spots on seminal plasma gels was similar from 15 to 18 weeks (45 and 47 spots; p > 0.05), increased until 24 weeks (141 spots) and 28 weeks (170 spots; p < 0.05) and remained without significant (p > 0.05) changes from 28 to 48 weeks (186 ± 10 spots). Furthermore, the intensity of selected spots on 2D maps increased (p < 0.05) between 15 and 28 weeks, which preceded or coincided with the main developmental changes in sperm motility and percentage of defective sperm in the ejaculates. These results will support future studies designed to characterize specific seminal plasma proteins whose expression relate to the development of testis, epididymis and accessory sex glands.     

Sexual behavior and seminal quality of Piau boars

The objective of this study was to assess parameters of sexual behavior, seminal characteristics, testicular measurements, and their correlations in Piau boars. A total of 169 observations of the sexual behavior and prelude duration (courting) of 10 Piau boars aged from 0.8 to 6.4 years were made; semen was also collected from these animals. The testicular length (TL), testicular width (TW), testicular shape (TS; 1-5), and testicular volume (TV) of these animals were measured. The seminal physical and morphological characteristics of 60 ejaculates from 6 boars were assessed for volume of ejaculate (VOL), volume of the gelatinous portion (VOLGEL), aspect (ASP), sperm motility (MOT), spermatic vigor (VIG), spermatic concentration per milliliter (CONCML), total concentration (CONCT), and morphological characteristics (major defects [MAJDEF]; minor defects [MINDEF]; and total defects [TDEF]). The animals were subdivided into 3 age groups: G1 (0.8-2.0 years old), G2 (2.1-3.6 years old), and G3 (3.7 and 6.4 years olds). The physiological signs observed included duration of prelude (12.1 ± 4.1 minutes), time to collect semen (5.2 ± 1.8 minutes), nasonasal or genital contact behavior (71.6%), saliva emission (85.2%), urination during prelude (10.1%), penile exposure before collection (26.6%), flank stimulus (81.7%), chewing behavior (95.3%), and biting of the female or dummy (74.6%). The mean values of TL, TW, and TV were 11.8 ± 2.2 cm, 6.2 ± 1.3 cm and 754.1 ± 283.5 cm³, respectively. The older animals had the highest mean values of testicular measurements (P < 0.05) but the lowest mean values of CONCML and CONCT (P < 0.05). All the animals presented similar patterns of ejaculates (P > 0.05); the mean values of VOL, VOLGEL, MOT, VIG, and CONCT were 149.1 ± 51.1 mL, 29.7 ± 10.0 g, 76.2% ± 9.5%, 3.3 ± 0.5, and 29.4 ± 19.5 × 10⁹ total sperm in the ejaculate, respectively. The prevalence rates of defects were 5.9% ± 2.6% for MAJDEF, 10.2% ± 4.0% for MINDEF, and 16.2% ± 5.5% for TDEF; all animals were classified as being sound for breeding. The assessed characteristics were similar to those observed in swine of commercial lineages and within the standards established by the Brazilian College of Animal Reproduction for the species.     

Seminal parameters and field fertility of cryopreserved donkey jack semen after insemination of horse mares

Reasons for performing study: As mule production is often concentrated in remote areas of the world, a simplified semen cryopreservation protocol is required. Aim: To compare the seminal parameters of cryopreserved donkey semen in lactose‐EDTA and lactose‐yolk extenders and the fertility rates on horse mares. Methods: Trial 1: Sperm total and progressive motility, vigour (scale 0–5), morphology (major and minor defects) and plasma membrane integrity (HOST) were evaluated in 25 ejaculates from 5 donkey jacks immediately after collection (raw), after chilling to 5°C (chilled) and after freezing/thawing. The semen was mixed with skimmed‐milk extender, centrifuged, and then re‐suspended in lactose‐EDTA or lactose‐yolk extender. Semen was loaded into 0.5 ml straws and chilled to 5°C for 1 h, after which samples were either evaluated (chilled semen) or placed above liquid nitrogen for 20 min prior to immersion. Seminal parameters were evaluated by ANOVA and Tukey's test. Trial 2: Cryopreserved semen from 3 males was used to inseminate 53 mares at 60 oestrous cycles randomly assigned to lactose‐yolk (n = 30 cycles) or lactose‐EDTA (n = 30 cycles) extenders. Pregnancy diagnosis was performed 15 and 25 days post ovulation. The pregnancy rates were compared using Chi‐squared tests. Results: Trial 1: No significant differences were evident in any seminal parameters between extenders after either chilling or cryopreservation. Total and progressive motility were significantly (P<0.05) lower in cryopreserved semen than raw and chilled semen for both extenders. Trial 2: Pregnancy rates did not significantly differ between extenders (lactose‐EDTA extender 53.33 and 43.33%; lactose‐yolk 50.0 and 46.66% for Days 15 and 25 post ovulation, respectively). Conclusions: Cryopreservation of donkey semen using the simplified lactose‐yolk extender resulted in similar seminal parameters and fertility rates when compared to lactose‐EDTA extender. Potential relevance: Lactose‐yolk extender may be advocated as a simple, easy to prepare extender, for use in geographically isolated enterprises producing mules throughout the world.     

Removal of Seminal Plasma Enhances Membrane Stability on Fresh and Cooled Stallion Spermatozoa

Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some ‘poor cooler’ stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4°C: motility, plasma membrane integrity as evaluated by hypo‐osmotic swelling test, acrosome integrity and response to a pharmacological induction of acrosome reaction using ionophore A23187. Sixty‐six ejaculates from 14 stallions were used, including stallions showing high or low sperm motility after cooled storage. Centrifugation without removal of seminal plasma did not affect sperm parameters. Removal of seminal plasma did not affect motility, but significantly stabilized sperm membranes, as demonstrated by a higher response to the osmotic challenge, and a reduced reactivity of the acrosome. Moreover, for the same semen sample, the response to an induction of acrosome reaction was significantly higher when the induction was performed in the presence of seminal plasma, compared with the induction in the absence of seminal plasma. This was observed both for fresh and cooled semen. When the induction of acrosome reaction with ionophore A23187 is used to evaluate sperm quality, care must therefore be taken to standardize the proportion of seminal plasma between samples. For the 10 stallions serving at least 25 mares, the only variable significantly correlated with fertility was motility. The influence of membrane stabilization regarding fertility requires further investigations.