Effect of spermatozoal concentration and number on fertility of frozen equine semen

Information on the number of motile spermatozoa needed to maximize pregnancy rates for frozen-thawed stallion semen is limited. Furthermore, concentration of spermatozoa per 0.5-mL straw has been shown to affect post-thaw motility (7). The objectives of this study were 1) to compare the effect of increasing the concentration of spermatozoa in 0.5-mL straws from 400 to 1,600 x 10(6) spermatozoa/mL on pregnancy rate of mares, and 2) to determine whether increasing the insemination dose from approximately 320 to 800 million progressively motile spermatozoa after thawing would increase pregnancy rates. Several ejaculates from each of 5 stallions were frozen in a skim milk-egg yolk based freezing medium at 2 spermatozoal concentrations in 0.5-mL polyvinyl-chloride straws. Half of each ejaculate was frozen at 400 x 10(6) cells/mL and half at 1,600 x 10(6) cells/mL. Insemination doses were based on post-thaw spermatozoal motility and contained approximately 320 x 10(6) (320 to 400) motile spermatozoa or approximately 800 x 10(6) (800 to 900) motile spermatozoa. Sixty-three mares were assigned to 1 of 4 spermatozoal treatments (1--low spermatozoal number, low concentration; 2--low spermatozoal number, high concentration; 3--high spermatozoal number, low concentration; 4--high spermatozoal number, high concentration) and were inseminated daily. Post-thaw spermatozoal motility was similar for cells frozen at both spermatozoal concentrations (P > 0.1). One-cycle pregnancy rates were 15, 40, 28 and 33%, respectively, for Treatments 1, 2, 3 and 4. Packaging spermatozoa at the high concentration tended to increase pregnancy rates vs packaging at the low concentration (37 vs 22%; P = 0.095). Furthermore, when the lower spermatozoal number was used, there tended (P < 0.1) to be a higher pregnancy rate if spermatozoa were packaged at the higher concentration. There was no increase in pregnancy rates when higher numbers of motile spermatozoa were inseminated (27 vs 31%; P > 0.1). Based on these results, a single 0.5-mL straw dose containing 800 x 10(6) spermatozoa should be used and each insemination dose should contain approximately 320 x 10(6) motile spermatozoa. Fertility trials utilizing other freezing extenders are necessary before recommending a single 0.5-mL insemination dose for all freezing extenders.     

Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen

In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 degrees C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. Group 3 mares were inseminated once with 2 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. All mares were given 2500 IU i.v. hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered i.m. 10 mg of PGF2 alpha and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 x 10(9) progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 x 10(6) motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 degrees C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 x 10(9) progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 x 10(9) progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 x 10(9) progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 x 10(9) progressively motile spermatozoa given on two consecutive days. However, a shorter interval (< or = 24 h rather than > 0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.     

Low dose insemination of mares using non-sorted and sex-sorted sperm.

Mares are generally inseminated with 500 million progressively motile fresh sperm and approximately 1 billion total sperms that have been cooled or frozen. Development of techniques for low dose insemination would allow one to increase the number of mares that could be bred, utilize stallions with poor semen quality, extend the use of frozen semen, breed mares with sexed semen and perhaps reduce the incidence of post-breeding endometritis. Three low dose insemination techniques that have been reported include: surgical oviductal insemination, deep uterine insemination and hysteroscopic insemination.Insemination techniques: McCue et al. [J. Reprod. Fert. 56 (Suppl.) (2000) 499] reported a 21% pregnancy rate for mares inseminated with 50,000 sperms into the fimbria of the oviduct.Two methods have been reported for deep uterine insemination. In the study of Buchanan et al. [Theriogenology 53 (2000) 1333], a flexible catheter was inserted into the uterine horn ipsilateral to the corpus luteum. The position of the catheter was verified by ultrasound. Insemination of 25 million or 5 million spermatozoa resulted in pregnancy rates of 53 and 35%, respectively. Rigby et al. [Proceedings of 3rd International Symposium on Stallion Reproduction (2001) 49] reported a pregnancy rate of 50% with deep uterine insemination. In their experiment, the flexible catheter was guided into position by rectal manipulation.More studies have reported the results of using hysteroscopic insemination. With this technique, a low number of spermatozoa are placed into or on the uterotubal junction. Manning et al. [Proc. Ann. Mtg. Soc. Theriogenol. (1998) 84] reported a 22% pregnancy rate when 1 million spermatozoa were inserted into the oviduct via the uterotubal junction. Vazquez et al. [Proc. Ann. Mtg. Soc. Theriogenol. (1998) 82] reported a 33% pregnancy rate when 3.8 million spermatozoa were placed on the uterotubal junction. Recently, Morris et al. [J. Reprod. Fert. 188 (2000) 95] utilized the hysteroscopic insemination technique to deposit various numbers of spermatozoa on the uterotubal junction. They reported pregnancy rates of 29, 64, 75 and 60% when 0.5, 1, 5 and 10 million spermatozoa, respectively, were placed on the uterotubal junction.Insemination of sex-sorted spermatozoa: One of the major reasons for low dose insemination is insemination of X- or Y-chromosome-bearing sperm. Through the use of flow cytometry, spermatozoa can be accurately separated into X- or Y-bearing chromosomes. Unfortunately, only 15 million sperms can be sorted per hour. At that rate, it would take several days to sort an insemination dose containing 800 million to 1 billion spermatozoa. Thus, low dose insemination is essential for utilization of sexed sperm. Lindsey [Hysteroscopic insemination with low numbers of fresh and cryopreserved flow-sorted stallion spermatozoa, M.S. Thesis, Colorado State University, Fort Collins, CO, USA, 2000] utilized either deep uterine insemination or hysteroscopic insemination to compare pregnancy rates of mares inseminated with sorted, fresh stallion sperm to those inseminated with non-sorted, fresh stallion sperm. Hysteroscopic insemination resulted in more pregnancies than ultrasound-guided deep uterine insemination. Pregnancy rate was similar for mares bred with either non-sorted or sex-sorted spermatozoa.In a subsequent study, Lindsey et al. [Proceedings of 5th International Symposium on Equine Embryo Transfer (2000) 13] determined if insemination of flow-sorted spermatozoa adversely affected pregnancy rates and whether freezing sex-sorted spermatozoa would result in pregnancies. Mares were assigned to one of four groups: group 1 was inseminated with 5 million non-sorted sperms using hysteroscopic insemination; group 2 was inseminated with 5 million sex-sorted sperms using hysteroscopic insemination; group 3 was inseminated with non-sorted, frozen-thawed sperm; and group 4 was inseminated with sex-sorted frozen sperm. Pregnancy rates were similar for mares inseminated with non-sorted fresh sperm, sex-sorted fresh sperm and non-sorted frozen sperm (40, 37.5 and 37.5%, respectively). Pregnancy rates were reduced dramatically for those inseminated with sex-sorted, frozen-thawed sperm (2 out of 15, 13%). These studies demonstrated that hysteroscopic insemination is a practical and useful technique for obtaining pregnancies with low numbers of fresh spermatozoa or low numbers of frozen-thawed spermatozoa. Further studies are needed to determine if this technique can be used to obtain pregnancies from stallions with poor semen quality. In addition, further studies are needed to develop techniques of freezing sex-sorted spermatozoa.     

Effect of insemination dose on pregnancy rate in mares

Different insemination doses have been used for artificial insemination(AI) in horses. Since the insemination dose can affect the pregnancy rate, it is important to ensure that an adequate dose be used regardless of the type of inseminationprotocol used. The aim of this study was to find out if it is possible to decrease the insemination dose from 500 x 10(6) progressively motile spermatozoa to 300 x 10(6) progressively motile spermatozoa and still maintain an acceptable pregnancy rate when using extended fresh semen. Thirteen stallions of known fertility and a well-defined group of 64 mares were used in the study. The mares were randomly assigned to 1 of 2 insemination groups. Examination for pregnancy was performed by ultrasonography per rectum approximately 16 d after the last insemination. When using an insemination dose of 300 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 75%. With an insemination dose of 500 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 64%. There was no significant difference in the pregnancy rate between the 2 insemination doses (P = 0.341). We conclude that when using fresh extended semen it is unlikely that an insemination dose of 300 x 10(6) progressively motile spermatozoa would yield a lower pregnancy rate than a dose of 500 x 10(6) progressively motile spermatozoa if stallions with good quality semen are selected.     

Homologous prostatic fluid added to frozen-thawed dog spermatozoa prior to intravaginal insemination of bitches resulted in better fertility than albumin-free TALP

The aim of this study was to determine whether canine prostatic fluid has intrinsic effects resulting in higher fertility than albumin-free Tyrode's albumin lactate pyruvate (afTALP) when added to thawed semen prior to intravaginal insemination. Twenty-four German shepherd bitches were inseminated intravaginally with frozen-thawed spermatozoa to which either homologous prostatic fluid (Group P; 12 bitches) or afTALP (Group T; 12 bitches) was added to give a final insemination volume of 7mL. Each bitch was inseminated daily starting when the vaginal folds first became angular and continuing until the day before a diestrus vaginal smear was first seen. Bitches were spayed about 3 weeks after the onset of diestrus and the number of corpora lutea and the number of conceptuses counted. Group P and Group T bitches were, respectively, inseminated 5.3+/-1.0 and 5.8+/-2.1 times with 48.9+/-8.6 and 50.4+/-8.3 million progressively motile spermatozoa per insemination. Eight Group P bitches and 10 Group T bitches conceived with totals of 76 and 45 conceptuses and 126 and 117 corpora lutea, respectively. Odds of conception were taken as the number of conceptuses divided by (the number of corpora lutea minus the number of conceptuses). After adjustment for the number of progressively motile spermatozoa per day and the random effect of bitch, the addition of prostatic fluid resulted in an increased odds of conception compared to afTALP. This effect decreased as the number of progressively motile spermatozoa per day increased.     

Sources of variation in the reproductive performance of ewes inseminated with frozen-thawed ram semen by laparoscopy

Fertility data from 8 artificial insemination programs, involving more than 5000 ewes and 110 rams in 3 flocks, were analyzed to determine variation due to individual AI program and ram in the reproductive performance of ewes inseminated with frozen-thawed semen by laparoscopy. The semen had been previously frozen by commercial AI centers in either pellets or straws. Both AI program and individual ram affected the proportion of ewes pregnant and the number of fetuses per ewe inseminated, but not the number of fetuses per pregnant ewe. Semen samples from 97 of the rams used were analyzed on a Hamilton Thorn HTM 2000 image analyzer for sperm concentration, percentage of motile and progressively motile spermatozoa, mean progressive velocity, and mean linear index. The correlations between these traits and reproductive performance obtained after insemination were calculated. There was large variation in the quantity and quality of the frozen semen, but only the number of total and motile spermatozoa inseminated per ewe was correlated with fertility (0.25 and 0.26, respectively). Regression analysis showed that none of the traits measured were useful for predicting fertility.     

Reproductive response of ewes synchronized with different lengths of MGA treatments in intrauterine insemination program

A total of 415 fat tailed ewes were randomly assigned to two groups to assess the effect of duration of melengestrol acetate (MGA) (9 versus 12d) administration on reproductive parameters associated with laparoscopic artificial insemination. At the end of MGA treatment, ewes in each group were subdivided and inseminated with one of two different insemination doses (10×10(7) or 20×10(7) sperm per 0.5 ml insemination dose) of fresh diluted semen. Inseminations were carried out 11-18 h after first detected estrus. Ewes were screened for their return to oestrus from 10 to 21 days post AI and inseminated at their returned oestrus. Pregnancy diagnosis was done from approximately 55 days after insemination in both synchronized and return estrus. For short (9-day) and long (12-day) term MGA treated groups, estrus rates were 62% versus 89% (P<0.0001), respectively. Ewes (n=115) that returned to estrus were inseminated (7-11h after estrus detection) with fresh diluted semen at different doses (20×10(7) or 40×10(7) or 60×10(7) sperm per 0.5 ml insemination dose). Pregnancy rates were 41% and 44% for short term and long term MGA treated ewes, respectively. Pregnancy rate of ewes which returned to oestrus was 53.4%. There was a significant (P<0.05) increase in pregnancy rates (38-52% for 11-16 h; 63% for 17-18 h) when insemination was held at 17-18 h after first detected estrus following MGA treatments. Pregnancy rates were found to be similar in ewes inseminated with 10×10(7) (36%) or 20×10(7) (47%) motile spermatozoa at first AI, and 20×10(7) (44%) or 40×10(7) (59%) or 60×10(7)(48%) at second AI. It was concluded that short term MGA treated ewes were recorded with lower estrus rates but was similar to pregnancy rates with long term MGA treatment. Acceptable pregnancy rates were achieved in MGA induced estrus when insemination is conducted at 17-18 h after estrus onset and with 20×10(7) sperm per insemination dose.     

Administration of oxytocin immediately after insemination does not improve pregnancy rates in mares bred by fertile or subfertile stallions

It is probable that reduced pregnancy rates in mares bred to subfertile stallions is attributable, in part, to the reduced number of normal spermatozoa that colonize the oviduct. Administration of oxytocin stimulates both uterine and oviductal contractility. The hypothesis that oxytocin may enhance sperm transport to/into the oviducts, and thereby increase pregnancy rates, was tested in 2 trials. For both trials, fertile estrous mares with follicles > or = 35 mm in diameter were inseminated once at 24 h after administration of 1500 to 2000 U hCG. The inseminate dose was limited to 100 million spermatozoa in order to lower pregnancy rates and thus increase the chance of detecting a treatment effect. Pregnancy status was determined by transrectal ultrasound examination 14 to 16 d after insemination. In Trial 1, 49 mares were inseminated with 4 mL extended semen from 1 of 3 stallions (1 fertile and 2 subfertile males). Immediately after insemination, the mares were administered either 20 U oxytocin or 1 mL saline intravenously. In Trial 2, 51 mares were inseminated with 4 mL extended semen from 1 of 4 stallions (1 fertile and 1 subfertile male used in Trial 1, and 2 additional fertile males). Immediately after insemination, and again 30 min later, mares were administered either 5 U oxytocin or 0.25 mL saline intramuscularly. To test for effects of treatment with oxytocin and for the interaction between semen quality and treatment, a generalized linear mixed regression model was used that accounted for the split-plot design (treatment within stallions), the random effect of stallion, the fixed effect of semen quality, the binary outcome of a single breeding trial, and the varying number of trials per stallion/treatment groups. Three treatment protocols or regimens were used: placebo, 5 U oxytocin injected twice intramuscularly, and 20 units oxytocin injected twice intravenously. Semen was classified as high (fertile stallions) or low (subfertile stallions) quality. No interaction between semen quality and treatment was detected (P > 0.10). The pregnancy rate of mares treated with oxytocin immediately after insemination was 30% (15/50) compared with 50% (25/50) for mares treated with saline immediately after breeding. Administration of oxytocin did not affect pregnancy rates (P > 0.10).     

Fertility comparison between breeding at 24 hours or at 24 and 48 hours after collection with cooled equine semen

It has become a common practice in the equine breeding industry to send 2 insemination doses for breeding with transported cooled semen, one to be used for the initial insemination upon arrival, and the other to be held a second insemination the next day. One fertile stallion and 36 fertile mares were used to determine if breeding once with 1 dose of semen cooled for 24 h would improve fertility compared with breeding twice, 1 d apart, with half the dose of semen cooled for 24 h on the first day of breeding and half cooled for 48 h on the second day of breeding. Mares were given two intramuscular injections of 10 mg PGF2 alpha 14 d apart. Following the second injection, mares were teased with a stallion and their ovaries were scanned by transrectal ultrasonography daily. When a dominant follicle (> 35 mm diameter) was detected, 1500 units hCG were injected intravenously, and the mares were inseminated. Semen was collected in advance of anticipated breeding, mixed in nonfat dry milk solids-glucose extender to a concentration of 25 million sperm/mL, and placed in 2 commercial cooling containers for 24 or 48 h of storage prior to breeding. Mares were randomly assigned to 1 of 2 insemination treatment groups: 1) Group T1 (n = 18), in which mares were inseminated on the day of hCG injection with 500 million spermatozoa cooled for 24 h, or 2) Group T2 (n = 18), in which mares were inseminated on the day of hCG injection with 250 million spermatozoa cooled for 24 h, and again on the following day with 250 million spermatozoa cooled for 48 h. Pregnancy status was confirmed by transrectal ultrasonographic examination at 14 and 16 d after ovulation. Pregnancy rates were the same for both insemination treatment groups (12/18; 67%). There was no advantage to holding half of the insemination dose for rebreeding on the following day.     

The effects of different insemination regimes on fertility in mares

This study investigated the effects of different artificial insemination (AI) regimes on the pregnancy rate in mares inseminated with either cooled or frozen-thawed semen. In essence, the influence of three different factors on fertility was examined; namely the number of inseminations per oestrus, the time interval between inseminations within an oestrus, and the proximity of insemination to ovulation. In the first experiment, 401 warmblood mares were inseminated one to three times in an oestrus with either cooled (500 x 10(6) progressively motile spermatozoa, stored at +5 degrees C for 2-4 h) or frozen-thawed (800 x 10(6) spermatozoa, of which > or =35% were progressively motile post-thaw) semen from fertile Hanoverian stallions, beginning -24, -12, 0, 12, 24 or 36 h after human chorionic gonadotrophin (hCG) administration. Mares were injected intravenously with 1500 IU hCG when they were in oestrus and had a pre-ovulatory follicle > or =40mm in diameter. Experiment 2 was a retrospective analysis of the breeding records of 2,637 mares inseminated in a total of 5,305 oestrous cycles during the 1999 breeding season. In Experiment 1, follicle development was monitored by transrectal ultrasonographic examination of the ovaries every 12 h until ovulation, and pregnancy detection was performed sonographically 16-18 days after ovulation. In Experiment 2, insemination data were analysed with respect to the number of live foals registered the following year. In Experiment 1, ovulation occurred within 48 h of hCG administration in 97.5% (391/401) of mares and the interval between hCG treatment and ovulation was significantly shorter in the second half of the breeding season (May-July) than in the first (March-April, P< or =0.05). Mares inseminated with cooled stallion semen once during an oestrus had pregnancy rates comparable to those attained in mares inseminated on two (48/85, 56.5%) or three (20/28, 71.4%) occasions at 24 h intervals, as long as insemination was performed between 24 h before and 12 h after ovulation (78/140, 55.7%). Similarly, a single frozen-thawed semen insemination between 12 h before (31/75, 41.3%) and 12 h after (24/48, 50%) ovulation produced similar pregnancy rates to those attained when mares were inseminated either two (31/62, 50%) or three (3/9, 33.3%) times at 24 h intervals.In the retrospective study (Experiment 2), mares inseminated with cooled semen only once per cycle had significantly lower per cycle foaling rates (507/1622, 31.2%) than mares inseminated two (791/1905, 41.5%), three (464/1064, 43.6%) or > or =4 times (314/714, 43.9%) in an oestrus (P< or =0.001). In addition, there was a tendency for per cycle foaling rates to increase when mares were inseminated daily (619/1374, 45.5%) rather than every other day (836/2004, 42.1%, P = 0.054) until ovulation.It is concluded that under conditions of frequent veterinary examination, a single insemination per cycle produces pregnancy rates as good as multiple insemination, as long as it is performed between 24 h before and 12 h after AI for cooled semen, or 12 h before and 12 h after AI for frozen-thawed semen. If frequent scanning is not possible, fertility appears to be optimised by repeating AI on a daily basis.     

Use of a semen extender containing antibiotic to improve the fertility of a stallion with seminal vesiculitis due to Pseudomonas aeruginosa

A breeding trial was conducted to determine if a semen extender containing polymixin-B sulfate would improve the fertility of a stallion with seminal vesiculitis due to Pseudomonas aeruginosa . Twenty-three mares were bred to the stallion by one of three methods: artificial insemination with raw semen (Group 1, n = 10), artificial insemination with semen mixed 1:1 with a nonfat dry skim milk/glucose extender containing 1000 units/ml polymixin-B sulfate (Group 2, n = 9), or natural service immediately following infusion of the uterus with 100 ml of the same extender (Group 3, n = 4). Artificial breedings contained a minimum insemination dose of 500 x 10(6) progressively motile spermatozoa. All mares were bred every other day while in estrus. Pregnancy status was determined by transrectal ultrasound examination 15 d after the last breeding. First-cycle pregnancy rate for Group 2 mares (78%) was greater (P < 0.01) than for Group 1 mares (10%). There was a tendency (P = 0.10) for the pregnancy rate of Group 3 mares (50%) to be greater than Group 1 mares. The use of a semen extender containing polymixin-B sulfate improved the fertility of this stallion.     

Fertility of frozen-thawed stallion semen extended in lactose-EDTA-egg yolk extender and packaged in 1.0-ml straws

The fertility of frozen-thawed and fresh semen from each of three stallions was compared in an experiment with a randomized block design using 128 mares. Semen was collected every third day, extended in lactose-EDTA-egg yolk extender at a concentration of 500 × 10⁶ progressively motile sperm per 1.0 ml, and frozen in individual-dose, 1.0-ml straws (1.9 mm × 267 mm). The same stallions were collected daily for inseminations with fresh semen. For each insemination dose with fresh semen, 300 × 10⁶ progressively motile sperm were added to 10 ml of heated skim milk extender. Mares were inseminated daily from the second day of estrus through the end of estrus. Of 52 ejaculates processed and frozen, 38% were discarded because < 35% of the sperm were progressively motile after thawing. Based on rectal palpations on day 50 post-ovulation, pregnancy rates for inseminations during one estrus to semen from the three stallions were 17, 33 and 35% for frozen-thawed semen and 60, 62 and 64% for fresh semen. Pregnancy rates with frozen semen from two of the three stallions were 54% of the rates attained with fresh semen.     

Insemination of mares with low numbers of either unsexed or sexed spermatozoa

Two experiments were conducted to determine pregnancy rates in mares inseminated 1) with 5, 25 and 500 x 10(6) progressively motile spermatozoa (pms), or 2) with 25 x 10(6) sex-sorted cells. In Experiment 1, mares were assigned to 1 of 3 treatments: Group 1 (n=20) was inseminated into the uterine body with 500 x 10(6) pms. Group 2 (n=21) and Group 3 (n=20) were inseminated into the tip of the uterine horn ipsilateral to the preovulatory follicle with 25 and 5 x 10(6) pms, respectively. Mares in all 3 groups were inseminated either 40 (n=32) or 34 h (n=29) after GnRH administration. More mares became pregnant when inseminated with 500 x 10(6) (18/20 = 90%) than with 25 x 10(6) pms (12/21 = 57%; P<0.05), but pregnancy rates were similar for mares inseminated with 25 x 10(6) vs 5 x 10(6) pms (7/20 = 35%) (P>0.1). In Experiment 2, mares were assigned to 1 of 2 treatments: Group A (n=11) was inseminated with 25 x 10(6) spermatozoa sorted into X and Y chromosome-bearing populations in a skimmilk extender. Group B (n=10) mares were inseminated similarly except that spermatozoa were sorted into the skimmilk extender + 4% egg yolk. Inseminations were performed 34 h after GnRH administration. Freshly collected semen was incubated in 224 microM Hoechst 33342 at 400 x 10(6) sperm/mL in HBGM-3 for 1 hr at 35 degrees C and then diluted to 100 x 10(6) sperm/mL for sorting. Sperm were sorted by sex using flow cytometer/cell sorters. Spermatozoa were collected at approximately 900 cells/sec into either the extender alone (Group A) or extender + 4% egg yolk (Group B), centrifuged and suspended to 25 x 10 sperm/mL and immediately inseminated. Pregnancy rates were similar (P>0.1) between the sperm treatments (extender alone = 13/10, 30% vs 4% EY + extender = 5/10, 50%). Based on ultrasonography, fetal sex at 60 to 70 d correlated perfectly with the sex of the sperm inseminated, demonstrating that foals of predetermined sex can be obtained following nonsurgical insemination with sexed spermatozoa.     

Effect of intrauterine treatment with prostaglandin E2 prior to insemination of mares in the uterine horn or body

Two trials were conducted to investigate the effects of intrauterine infusion of PGE2 and uterine horn insemination on pregnancy rates in mares achieved by breeding with a suboptimal number of normal spermatozoa. Estrus was synchronized and mares were teased daily with a stallion to detect estrus. Mares in estrus were examined by transrectal palpation and ultrasonography to monitor follicular status. On the first day a 35-mm diameter follicle was present, hCG (1500 IU, iv) was administered and the mares were bred the next day. Mares (Trial 1, n = 34; Trial 2, n = 28) were inseminated with 25 million total spermatozoa from either a stallion with good semen quality (Trial 1) or poor semen quality (Trial 2). In each trial, mares were assigned to 1 of 4 treatment groups as follows: Group PGE-HI - infusion of 0.25 mg PGE2 into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the proximal end of the same uterine horn; Group PGE-BI - infusion of 0.25 mg PGE2 into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the uterine body; Group SAL-HI - infusion of 1 mL sterile saline into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the proximal end of the same uterine horn; or Group SAL-BI - infusion of 1 mL sterile saline into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the uterine body. After breeding, mares were examined daily by transrectal ultrasonography to confirm ovulation, and were re-examined 14 to 16 d after ovulation for pregnancy status. Data were analyzed by Chi-square. Overall pregnancy rates were 59% for stallion 1 and 29% for stallion 2. Group pregnancy rates did not differ for mares bred by either stallion (P > 0.10). Pregnancy rates were not altered by horn insemination for either stallion (P > 0.10). Intrauterine infusion of PGE2 improved pregnancy rate in mares bred by the stallion with good quality semen (P < 0.05), but did not alter pregnancy rate in mares bred by the stallion with poor quality semen (P > 0.10). Further research is warranted to determine if intrauterine infusion of PGE2 will enhance spermatozoal colonization of the oviduct and pregnancy rates in mares, and if PGE-treatment will improve pregnancy rates achieved by subfertile stallions.     

Equine spermatozoal motility and fertility associated with the incorporation of d-(+)-mannose into semen extender

Mannose is capable of decreasing bacterial attachment to the uterine mucosa in mares. Bacteria gain entry into the mare's uterus during breeding; therefore, a practical method to deliver mannose to the uterus is to incorporate it into semen extenders. The effect of mannose on spermatozoal motility and subsequent sperm fertilizing capability is unknown. The present study evaluated progressive spermatozoal motility in semen extender formulations incorporating mannose and assessed the fertility of mares inseminated with a mannose-containing semen extender. In Experiment 1, progressive spermatozoal motility in extender mixtures containing 0 mannose (control), 25, 37 or 49 mg/mL mannose was evaluated at 20 degrees C or 5 degrees C holding temperatures for 0, 12, 24 and 48 h post-dilution. Measures were repeated three times using five stallions of proven fertility. High concentrations of mannose in the extender affected progressive motility beyond the time and temperature effects noted in the controls. Extender containing only mannose sugar (49 mg/mL) displayed an immediate depression in progressive motility compared with controls (45.5% versus 62.9%, respectively; P<0.001). The 37 mg/mL mannose extender had a less dramatic decrease in motility (P<0.05) and only after storage at 5 degrees C for > or =12h (48.7% versus 58.0%, respectively). Extender with 25 mg/mL mannose performed no differently than the control formulation under all conditions. In Experiment 2, two groups of mares (n=11 each) were inseminated with 500 x 10(6) progressively motile spermatozoa extended in a traditional skim milk (control) extender or the 37 mg/mL mannose extender preparation. A single-cycle pregnancy rate of 72% was achieved by both groups. Present data suggest that a semen extender containing up to 37 mg/mL mannose could maintain motile spermatozoa for on-farm use and 25 mg/mL mannose concentrations preserved motility during long-term cooling. Likewise, sperm extended with up to 37 mg/mL of mannose had the same fertilizing capability as sperm in traditional extender mixtures.     

Effect of Timing of Frozen Semen Insemination on Pregnancy Rate in Mares

Thirty-four mares were inseminated with frozen semen from one stallion during 2 oestrous cycles, every 48 h until ovulation took place and within 12 h after ovulation. Semen was frozen using the Colorado method. The insemination dose was from 200 to 400×106 progressively motile spermatozoa. Ovaries were examined every 12 h to determine time of ovulation. Examination for pregnancy was carried out using ultrasonography, 15 days after ovulation. Thirty-five per cent of mares inseminated < 24 h and 23% of mares inseminated between 24 - 48 h before ovulation were pregnant (p = 0.388). The pregnancy rate in all mares inseminated before ovulation was 30%. In the mares inseminated within 12 h of ovulation, it was 18% (p = 0.253). Younger mares (aged 4-10 yr) had a higher pregnancy rate (59%) than older mares (aged 11-15 yr) (23%), but the difference was not statistically significant (p = 0.057).     

The relationship of semen quality to pregnancy rate and litter size following artificial insemination in the bitch

Artificial insemination of 31 bitches with fresh, undiluted, semen resulted in 22 31 conceptions (71%) and the birth of 124 puppies. Inseminations using at least 220 x 10(6) spermatozoa of normal morphology resulted in a mean pregnancy rate of 81.5% (22 27 ). The pregnancy rates were significantly influenced by the total number of progressively motile or morphologically normal sperm cells per ejaculate (P < 0.05). The pregnancy rate was not influenced by the percentage of progressively motile or morphologically normal sperm cells per ejaculate (P > 0.1). Litter size was not influenced by sperm motility or morphology (P > 0.1).     

Adaptation of the hypoosmotic swelling test to assess functional integrity of stallion spermatozoal plasma membranes

Hypoosmotic swelling (HOS) is used for assessing plasma membrane function and fertilizing capacity of human spermatozoa. However, HOS solutions and methodologies have not been evaluated specifically for assessing stallion spermatozoa. The objective of this study was to identify a HOS solution and assay conditions specifically for stallions that would maximize spermatozoal plasma membrane swelling. The HOS solutions and assay conditions, including incubation time (15 to 180 min), temperature (25 degrees vs 37 degrees C), and total number of cells examined (100, 200 or 500) were evaluated. Assay consistency, accuracy, reliability and repeatability also were determined. Maximum spermatozoal plasma membrane swelling was observed in a 100 mosmol sucrose solution (P < 0.001). Incubation time (P = 0.67), temperature (P = 0.70) and total number of spermatozoa examined (P = 0.38 and P = 0.24 for 100 vs 200 and 100 vs 500, respectively) did not influence percent of HOS positive spermatozoa observed. A high degree of assay accuracy was indicated when a correlation of r = 0.998 was obtained between the HOS positive spermatozoa observed and expected when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. Assay consistency was demonstrated, as reflected by a mean coefficient of variation of 0.073 for 4 stallions. Also, the coefficient of variation from 2 analyses of variance was 0.168 and 0.096, indicating reasonably good assay reliability; estimates of repeatability from the same analyses were 0.794 and 0.968. The HOS test adapted to stallion spermatozoa in this study is a simple, highly accurate and consistent assay with good reliability and repeatability. Results observed under the conditions evaluated also permit some flexibility in adapting this assay to individual laboratory and practice settings for evaluating stallion spermatozoal plasma membranes.     

Selected times of insemination with microencapsulated bovine spermatozoa affect pregnancy rates of synchronized heifers

This experiment was designed to test whether spermatozoa encapsulated in an alginate poly-L-lysine matrix had an extended fertile life in vivo after insemination. Estrus was synchronized in 417 primiparous Friesian and Jersey heifers with a system based on a CIDR-B intravaginal device before the heifers were inseminated either during proestrus (24 h after device removal) or at estrus (48 h after device removal). Pregnancy rates to first inseminations did not differ between the 24 and 48 h inseminations (61 vs 60.6%) with liquid semen diluted in Caprogen (control) but differed with encapsulated semen (45.1 vs 68.6%). The difference in pregnancy rates between the 2 types of semen was more pronounced (P < 0.08) in the animals that were visually detected in estrus. The mean survival time of spermatozoa in the female reproductive tract following insemination at the 24-h insemination time was estimated to be 50 +/- 7.5 h. The increased pregnancy rate with insemination of encapsulated spermatozoa at 48 h could have been due to this process predisposing spermatozoa to capacitate soon after insemination.     

Influence of sperm number and seminal plasma on fertility of progestagen-treated sheep in confinement

Progestagen-impregnated vaginal sponges + PMSG were used to synchronize oestrus in crossbred adult ewes which were inseminated 56 h after sponge removal with 0.5 ml diluted semen containing 400, 200, 100, 50 or 25 x 10(6) spermatozoa per insemination. The diluent was skim milk-citrate or pooled seminal plasma. There was no difference in reproductive performance due to the insemination medium. Fertility (no. of ewes lambing) after insemination of 400 or 200 x 10(6) spermatozoa was 68% and was similar to that observed after natural service at progestagen-induced oestrus. When less than or equal to 100 x 10(6) spermatozoa were inseminated, fertility fell markedly and the number of lambs per ewe inseminated decreased. A decrease in litter size also occurred. The data indicate that insemination of 200 x 10(6) spermatozoa, i.e. less than 10% of the number in a single ram ejaculate, allows normal conception rates in progestagen-treated ewes.