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.     

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.     

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.     

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).     

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.     

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.     

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.     

Motility and fertility of equine spermatozoa in a milk extender after 12 or 24 hours at 20 degrees C

The effects of extender and storage at 20 degrees C on equine spermatozoa were evaluated in two experiments using embryo recovery as the end point. In both experiments, inseminations were every other day, starting on Day 2 or 3 of estrus or after a 35-mm follicle was detected, with 250 x 10(6) progressively motile cells (based on initial evaluation). In Experiment 1, semen from two stallions was used to compare the motility and fertility of spermatozoa maintained in a) heated skim milk extender at 37 degrees C with insemination in <1 h; b) E-Z Mixin extender at 37 degrees C with insemination in <1 h; and c) E-Z Mixin extender at 37 degrees C with cooling to 20 degrees C and insemination after storage for 12 h at 20 degrees C. The percentage of motile spermatozoa was 34% after 12 h compared to 55% at 0 h (P < 0.05). However, the percentage of mares from which an embryo was recovered 6.5 d after ovulation was 62, 56, and 50% for Treatments A, B, and C (P > 0.05). In Experiment 2, semen from three stallions was used to compare the motility and fertility of spermatozoa in a) E-Z Mixin extender at 37 degrees C with insemination in <1 h or b) E-Z Mixin extender at 37 degrees C with cooling to 20 degrees C and insemination after storage for 24 h at 20 degrees C. The percentage of motile spermatozoa was 17% after 24 h compared to 54% at 0 h (P < 0.05). There was no difference between treatments (P > 0.05) in the percentage of mares from which an embryo was recovered 6.0 d after ovulation (68 vs 62%) or among stallions. Thus, stallion semen extended in E-Z Mixin was held at 20 degrees C for 24 h without a marked decline in fertility.     

Effect of insemination timing on the fertilizing capacity of frozen/thawed equine spermatozoa

A breeding trial was conducted to evaluate the effect of insemination timing on the fertility of mares bred with frozen/thawed equine semen. One stallion and 60 reproductively sound, estrous-synchronized mares were included in the study. Mares were assigned to one of three groups (n = 20): 1) insemination with fresh semen every other day during estrus from detection of a 35-mm follicle until ovulation, 2) insemination with frozen/thawed semen every day during estrus from detection of a 35-mm follicle until ovulation or 3) insemination with frozen/thawed semen once, within 6 h after ovulation. Single-cycle 18-d pregnancy rates resulting from insemination with fresh semen (70%), preovulation insemination with frozen/thawed semen (60%) and postovulation insemination with frozen/thawed semen (55%) were not different (P > 0.05). Possibly, equivalent pregnancy rates could be achieved with frozen/thawed semen using either daily inseminations until ovulation occurs or frequent ovarian palpations with a single post-ovulation insemination. Further studies regarding the effect of insemination timing on stallion fertility are needed since the present investigation included only one stallion and a small number of mares.     

Stallion spermatozoa selected by single layer centrifugation are capable of fertilization after storage for up to 96 h at 6°C prior to artificial insemination

ABSTRACT: BACKGROUND: One of the challenges faced by equine breeders is ensuring delivery of good quality semen doses for artificial insemination when the mare is due to ovulate. Single Layer Centrifugation (SLC) has been shown to select morphologically normal spermatozoa with intact chromatin and good progressive motility from the rest of the ejaculate, and to prolong the life of these selected spermatozoa in vitro. The objective of the present study was a proof of concept, to determine whether fertilizing ability was retained in SLC-selected spermatozoa during prolonged storage. FINDINGS: Sixteen mares were inseminated with SLC-selected sperm doses that had been cooled and stored at 6°C for 48 h, 72 h or 96 h. Embryos were identified in 11 mares by ultrasound examination 16-18 days after presumed ovulation. CONCLUSION: SLC-selected stallion spermatozoa stored for up to 96 h are capable of fertilization.     

Effects of different artificial insemination techniques and sperm doses on fertility of normal mares and mares with abnormal reproductive history

The effects of different artificial insemination (AI) techniques and sperm doses on pregnancy rates of normal Hanoverian breed mares and mares with a history of barrenness or pregnancy failure using fresh or frozen-thawed sperm were investigated. The material included 187 normal mares (148 foaling and 39 young maiden mares) and 85 problem mares with abnormal reproductive history. Mares were randomly allotted into groups with respect to AI technique (routine AI into the uterine body, transrectally controlled deep intracornual AI ipsilateral to the preovulatory follicle, or hysteroscopic AI onto the uterotubal junction ipsilateral to the preovulatory follicle), storage method of semen (fresh, frozen-thawed), AI volume (0.5, 2, 12 ml), and sperm dose (50 x 10(6) or 300 x 10(6) progressively motile sperm (pms) for fresh semen and 100 or 800 x 10(6) frozen-thawed sperm with >35% post-thaw motility). The mares were inseminated once per cycle, 24 h after hCG administration when fresh semen was used, or 30 h for frozen-thawed semen. Differences in pregnancy rates between treatment groups were analyzed by Chi-squared test, and for most relevant factors (insemination technique, mare, semen, and stallion) expectation values and confidence intervals were calculated using multivariate logistic models. Neither insemination technique, volume, sperm dose, nor mare or stallion had significant effects (P > 0.05) on fertility. Type of semen, breeding mares during foal heat, and an interaction between insemination technique, semen parameters, and mares did have significant effects (P < 0.05). In problem mares, frozen semen AI yielded significantly lower pregnancy rates than fresh semen AI (16/43, 37.2% versus 25/42, 59.5%), but this was not the case in normal mares. In normal mares, hysteroscopic AI with fresh semen gave significantly (P < 0.05) better pregnancy rates than uterine body AI (27/38, 71% versus 18/38, 47.3%), whereas in problem mares this resulted in significantly lower pregnancy rates than uterine body AI (5/15, 33.3% versus 16/19, 84.2%). Our results demonstrate that for problem mares, conventional insemination into the uterine body appears to be superior to hysteroscopic insemination and in normal mares, the highest pregnancy rates can be expected by hysteroscopic insemination.     

Perceptions of obesity in a UK leisure-based population of horse owners

Pregnancy rates with cooled equine semen can be unsatisfactory and show great variation. Information about first cycle pregnancy rates and pregnancy rates per cycle are often lacking from publicly available records. This retrospective cohort study was performed to evaluate the fertility of the Norwegian Coldblooded trotter. The aim of the study was to compare the breeding results after insemination with fresh, extended with those of cooled, shipped semen among Norwegian Coldblooded trotter mares. First cycle pregnancy rate was the main parameter used to measure fertility. Stud-books were collected from four studs from the years 2006–2010. Statistical analyses were done in Stata using Chi square test and multivariable analyses where different models were compared based on Akaike’s information criterion. First cycle pregnancy rate, seasonal pregnancy rate and foaling rate all showed significant differences (P < 0.0001) when comparing mares inseminated at stud with mares inseminated with cooled, shipped semen, favoring artificial insemination (AI) at stud. First cycle pregnancy rate was 55.1 % for mares inseminated at stud with fresh extended semen and 42.2 % for mares inseminated with cooled shipped semen. The overall pregnancy rate per cycle was 84.4 % for AI at stud and 66.9 % for cooled, shipped semen. The parameters stud, mare age, number of inseminations within an estrus cycle and individual stallion were also investigated for influence on fertility. Few retrospective studies include the parameter of first cycle pregnancy rates. Our study does not differ dramatically when comparing seasonal pregnancy rates and foaling rates with similar studies. Fertility parameters for the Norwegian Coldblooded trotter do not differ significantly from most other studies of Coldblooded mares and other mare breeds around the world. But the difference in fertility parameters between AI at stud to AI with cooled semen between our study and others, indicates that higher pregnancy rates in Norwegian Coldblooded trotter may be possible.     

Sperm-induced leukocytosis in the equine uterus

The objective of this study was to investigate the inflammatory reaction induced in the equine uterus by insemination with fresh and frozen semen. Eleven groups (6 to 8 mares per group) were studied during 2 breeding seasons. The mares were inseminated using raw semen, frozen semen, extended fresh and frozen semen, concentrated fresh semen, seminal plasma and seminal extenders only. One group was bred naturally. Six hours after insemination, the uteri were flushed with 50 ml of phosphate-buffered saline (PBS). Seventeen out of 104 samples (16%) exhibited slight bacterial growth. Neutrophil concentrations were significantly (P < 0.05) higher in all treated mares than in the controls. Mares infused with PBS, seminal extenders or the supernatant from centrifuged frozen-thawed semen exhibited only a mild neutrophil response. Insemination with frozen semen resulted in higher neutrophil concentrations than insemination with extended fresh semen (means of 59 vs 5 million neutrophils/ml; P < 0.05). Highest neutrophil counts were found after insemination with frozen semen or concentrated fresh semen. Bacterial contamination of uteri was insignificant 6 hours after breeding. Neutrophilia seems to be induced by spermatozoa rather than bacteria. The intensity of the neutrophil reaction seems to depend on concentration and/or volume of inseminate.     

Insemination results with slow-cooled stallion semen stored for 70 or 80 hours

An insemination trial was conducted to evaluate the fertility of extended slow-cooled stallion spermatozoa stored for 70 h or 80 h at 5 to 7 degrees C before insemination. Then, 1 or 2 of the first sperm-rich fractions were collected with an open-ended vagina from 4 stallions. Semen from each stallion was diluted within 2 to 3 min after collection with a modified Kenney skim milk extender (6). The proportion of raw semen in the insemination doses was 24+/-6%. One insemination dose (25 to 50 ml) consisted of approximately 2 billion total spermatozoa. In the trial, palpation per rectum and ultrasonography of 34 mares (40 cycles) were performed every 12 h. The pregnancy rate per cycle (30-d) with semen stored for 70 h before insemination was 77% (17 cycles) and, with semen stored for 80 h, 57% (23 cycles). The difference was not statistically significant. The combined pregnancy rate per cycle was 65%. These results indicate that stallion semen can retain its fertilizing capacity for up to 80 h when collected and diluted using this procedure and when the inseminations are done less than 12 h after ovulation.     

The effect of insemination volume on pregnancy rates of pony mares

It has recently been reported that large insemination volumes might affect fertility of mares. The results from these studies are confounded by other factors, however, such as inadequate number of spermatozoa in the inseminate. We conducted a study to test whether volume alone affects fertility when sufficient numbers of spermatozoa are present. Semen from one stallion was collected, extended at 50 x 10(6) spermatozoa/ml, and stored in a commercial semen cooling device for 18 to 30 h before insemination. Ten pony mares were assigned during estrus in random pairs to be bred every other day with either 30 or 120 ml of extended cooled semen. Pregnancy was diagnosed by ultrasonography per rectum on Days 11, 12 and 13 after ovulation. On Day 13, the mares were given a luteolytic dose (5 mg) of PGF(2alpha). On the subsequent cycle, the mares were given the alternate treatment. The pregnancy rates in the 30- and 120-ml insemination volume groups were 7 9 and 10 10 , respectively; this difference was not significant (P=0.2). Embryonic growth from Day 11 to Day 13 was not different (P>0.05) between groups. We conclude that insemination volumes as large as 120 ml have no adverse effect on fertility.     

Insemination Results with Slow-Cooled Stallion Semen Stored for approximately 40 Hours

Semen from 3 stallions was extended using 2 methods (Kenney extender and a modified Kenney extender), slowly cooled, and stored for 41 ± 6 (s.d.) h before insemination. An insemination dose (40 ml) contained 1.5-2 billion spermatozoa. In the experiment, 26 mares were inseminated in 30 cycles. The pregnancy rate per cycle obtained with sperm stored in the Kenney extender was 87% (n=15). When the semen was extended with the modified extender, centrifuged and stored, the pregnancy rate was 60% (n=15). Inseminations were done every other day until ovulation was detected. If a mare ovulated more than 24 h after the last insemination, she was inseminated also after ovulation. The single-cycle pregnancy rate was 58% when the mares were inseminated only before ovulation (n=19) but the rate was 100% when the inseminations were done both before and after ovulation (n=9) or only after ovulation (n=2). The difference in pregnancy rates was significant (p<0.05), indicating that postovula-tory inseminations probably serve to ensure the pregnancies. The extending and handling methods used in this study resulted in a combined pregnancy rate of 73%, and appear thus to be useful for storing stallion semen for approximately 2 days.     

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).     

Insemination time and dilution rate of cooled and chilled ram semen affects fertility

Adult Merino ewes (n=448) were apportioned into two groups and inseminated with: extended at 30 degrees C with skim milk and stored for 6h at 15 degrees C (cooled semen) or extended with skim milk-citrate trisodium with egg yolk and stored for 24h at 5 degrees C (chilled semen). Each group was further subdivided according to the time of cervical insemination at 42, 46 and 50h after pessary (MAP-60 mg) removal and according to the dilution of the semen (120 x 10(6) spermatozoa in 0.05, 0.1 and 0.2 ml). The pregnancy rate after insemination with cooled semen was 50% better than that after chilled semen (56.7 vs. 37.5%; P<0.001). Pregnancy rate was not affected by the volume of insemination; however, there was a tendency of increased lambing rate with an insemination dose of 0.1 cc (1:2, dilution), especially when the ewes were inseminated with cooled semen. The effect of time on insemination was significant only in ewes inseminated with chilled semen at 5 degrees C (P<0.01). Insemination carried out 46 h after pessary removal resulted in higher pregnancy and lambing rate (36.5, 31.1; 52.0, 45.3; and 24.0, 20.0 at 42, 46 and 50h, respectively). Pregnancy of ewes inseminated with chilled semen at 46 h after pessary removal was similar to that obtained using cooled semen (52.0 vs. 56.7%). From this study, it is concluded that advancing the time of insemination with chilled semen at 5 degrees C improves pregnancy and that the lambing obtained under these conditions is similar to the one obtained with cooled semen.     

Pregnancy rates in mares following hysteroscopic or transrectally-guided insemination with low sperm numbers at the utero-tubal papilla

This study was conducted to evaluate two methods for insemination of a low number of sperm in the tip of the uterine horn, and to determine whether prebreeding intrauterine treatment with prostaglandin E(2) would improve pregnancy rates. Estrus was synchronized in 36 fertile Quarter Horse and Thoroughbred broodmares. When a dominant follicle >or=33 mm diameter was present, mares were treated with 2500 units hCG intravenously and were assigned to one of four treatment groups for insemination with five million total sperm in 200 microl extender the next day as follows: (1) Group PGE-HYS (n=9): 0.25mg PGE(2) in 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to hysteroscopic-guided inseminate placement onto the oviductal papilla; (2) Group SAL-HYS (n=9): 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to hysteroscopic-guided inseminate placement onto the oviductal papilla; (3) Group PGE-PIP (n=9): 0.25mg PGE(2) in 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to transrectally-guided pipette placement of the inseminate into the tip of the uterine horn; and (4) Group SAL-PIP (n=9): 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to transrectally-guided pipette placement of inseminate into the tip of the uterine horn. Mares in estrus were evaluated daily by transrectal ultrasonography to monitor follicular status and confirm ovulation. If mares had not ovulated within 2 days of insemination, the assigned treatment was repeated. Pregnancy status was evaluated by transrectal ultrasonography 12-14 days postovulation, and pregnancy rates were compared.No interaction between prebreeding treatment (SAL:PGE) and insemination protocol (HYS:PIP) on pregnancy rates occurred (P>0.10). Pregnancy rates did not differ between mares inseminated by HYS (12/18; 67%) or PIP (10/18; 56%) (P>0.10). Pregnancy rates did not differ between mares treated prior to breeding with PGE (11/18; 61%) or SAL (11/18; 61%) (P=1.00). In summary, satisfactory pregnancy rates were obtained when a low number of sperm were either placed directly onto the oviductal papilla using hysteroscopy or placed in the tip of the uterine horn using a transrectally-guided uterine pipette. Infusion of 0.25mg PGE(2) in the tip of the uterine horn 2h prior to insemination did not improve pregnancy rates.     

Synchronization of oestus and timed insemination of mares.

Oestrus was synchronized in 116 mares by means of an i.m. injection of prostaglandin F-2 alpha (Day 0) and of fluprostenol (a PG analogue) on Day 16. Mares were then randomly divided into three groups. Group A mares (N = 30) were given 2500 i.u. hCG I.M. ON Day 20 and artificially inseminated on Day 21 without detection of oestrus. Group B mares (N = 32) were given 2500 i.u. hCG i.m. on Day 20 and inseminated on Days 21 and 23, also without oestrus detection. Group C mares (N = 54) were teased on Days 18, 19, 21, 23 and 25 and inseminated on Days 19, 21, 23 and 25 while they were in oestrus. Semen was collected by artificial vagina from 3 stallions. One-third of the mares in each group were assigned to each stallion at random. The gel-free fraction was divided equally among the mares, and used within 1 h of collection. Pregnancy rates at about 60 days of gestation were not significantly different. A high rate of synchronization of oestrus (80%) was attained within 48 h of treatment with fluprostenol.