Successful artificial insemination for indoor breeding in the Japanese monkey (Macaca fuscata) and the cynomolgus monkey (Macaca fascicularis)

Methods of artificial insemination (AI) for indoor breeding in the Japanese monkey and the Cynomolgus monkey were investigated. For the Japanese monkey AI was carried out in six females during the winter mating season and in six females during the summer non-mating season. During the mating season, semen was inseminated near ovulation time in natural menstrual cycles. In the mating season study, three females inseminated at the uterine cavity became pregnant. Three inseminated at the cervical canal failed to become pregnant. For the non-mating season study, ovulation was induced artificially by PMSG and hCG and AI was carried out near the induced ovulation time. In the non-mating season, no animals became pregnant. Of four Cynomolgus monkeys used, pregnancy occurred in two animals inseminated near ovulation time in natural menstrual cycles. AI occurred at the uterine cavity in one and cervical canal in the other. In both species ovulation was verified by laparoscopy. Semen was collected by penile electro-stimulation then diluted to 2.5 to 5.0×10⁷/ml with Whitten's medium. Diluted semen of 0.2l was inseminated at the uterine cavity or cervical canal. Our results indicate the usefulness of vaginal AI as a method of artificial indoor breeding.     

Development of an artificial insemination protocol in llamas using cooled semen

The objective of this study was to design an AI protocol using cooled semen to obtain pregnancies in the llama. Each raw ejaculate was subdivided into four aliquots which were extended 1:1 with: (1) 11% lactose-egg yolk (L-EY), (2) Tris-citrate-fructose-egg yolk (T-F-EY), (3) PBS-llama serum (S-PBS) and (4) skim milk-glucose (K). Each sample reached 5°C in 2.5 h and remained at that temperature during 24 h. Percentages of the semen variables (motility, live spermatozoa) in ejaculates and samples cooled with L-EY were significantly greater than those obtained when cooling with the other extenders; therefore this extender was used (1:1) for all inseminations. Females were randomly divided into four groups (A-D) according to insemination protocol. Group A: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa kept at 37°C. Group B: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa, cooled to 5°C and kept for 24 h. Group C: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C and kept for 24 h. These groups (A-C) were inseminated between 22 and 24 h after induction of ovulation. Group D: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C, kept for 24 h and AI was carried out within 2 h after ovulation. Pregnancy rates were 75%, 0%, 0% and 23% for groups A, B, C and D respectively. These results indicate that it is possible to obtain llama pregnancies with AI using cooled semen and that the success of the technique would depend on the proximity to ovulation.     

Semen-induced ovulation in the bactrian camel (Camelus bactrianus)

Bactrian camels (63 female female, 8 male male) were used in the breeding season to determine the factors that will induce ovulation. After insemination of semen samples into the vagina, the ovaries were checked for ovulation by rectal palpation. The results indicated that ovulation was induced by the seminal plasma, but not by the spermatozoa, and the incidence of ovulation after insemination was 87%. Most of the females (66%) had ovulated by 36 h after insemination and the rest by 48 h, as after natural service. The least amount of semen required to elicit ovulation was about 1.0 ml. Intramuscular injections of LH, hCG and LHRH also caused ovulation, even in females that had not ovulated in response to insemination.     

Successful artificial insemination in the koala (Phascolarctos cinereus) using extended and extended-chilled semen collected by electroejaculation

Artificial insemination in the koala using chilled, electroejaculated semen provides for a marked improvement in the reproductive and genetic management of captive koala colonies in Australia and internationally, and makes available the option of using semen collected from wild populations to expand restricted gene pools. Dilution of koala semen for artificial insemination is complicated because koalas are induced ovulators, and it is thought that ovulating factors are present in the semen, so that semen extension for preservation purposes might be anticipated to result in a failure to induce ovulation. The first two experiments of this study were designed to determine whether artificial insemination using undiluted, extended, and extended-chilled semen collected by electroejaculation was capable of inducing a luteal phase and/or the production of pouch young. In Experiment 1, 1 ml undiluted electroejaculated semen, 2 ml diluted (1:1) semen, and 1 ml diluted (1:1) semen resulted in seven of nine, six of nine, and six of nine koalas showing a luteal phase, respectively; four pouch young were produced in each treatment. A second artificial insemination experiment was conducted in which 2 ml diluted (1:1) semen was administered in three groups of nine koalas. The first group received semen that had been collected and diluted immediately without chilling, the second group was deposited with semen stored chilled for 24 h, and the final group received semen that had been chilled for 72 h. In the first group, five females had a luteal phase, but none became pregnant. In group 2, two of the five females that had a luteal phase gave birth, whereas in group 3, four of the six females that had a luteal phase produced pouch young. In addition, experiment 3 was conducted to determine whether it was possible to produce pouch young by naturally mating koalas that were in the latter stages of their behavioral estrus; this information is important to the logistics of transporting koala semen for artificial insemination by establishing the maximum time frame in which females might be expected to shed a fertile oocyte. Of the 12 females mated on Day 8 of estrus, 6 gave birth, whereas only 3 of the 10 females naturally mated on Day 10 of estrus produced pouch young. The majority of females (21 of 22) in experiment 3 showed evidence of a luteal phase. Together, these experiments have shown that it is possible to use undiluted, extended, or extended-chilled semen to produce koala offspring up to Day 8 of estrus at conception rates similar to those achieved following natural mating. These findings represent a significant advancement in the use of reproductive technology in marsupials and provide the basis for the shipment of koala semen over long distances. The pouch young produced in this study represent the first marsupials born following artificial insemination of extended-chilled semen and bring the total number of koalas produced by artificial insemination to 31.     

Distribution of spermatozoa in the female reproductive tract of the domestic cat in relation to ovulation induced by natural mating

The purposes of this study were to demonstrate the localization of spermatozoa in the reproductive tract of female domestic cats before (30 min and 3 h after mating) and after ovulation (48 and 96 h after mating), and to evaluate the efficiency of two techniques for studying sperm distribution. Estrus was induced in twenty-four female cats using 100 IU eCG and the females were divided into four groups with six females per group. The same male cat was used for mating with all the females. One group of six females was mated once; the others were mated four times in 1 h. Ovariohysterectomy was performed at 30 min, 3 h, 48 h, and 96 h after mating and the excised reproductive tracts were divided into seven segments on each side: infundibulum, ampulla, isthmus, uterotubal junction (UTJ), cranial and caudal uterine horn, and uterine body. The vagina and the lumina of the segments from one side were flushed with 0.5 ml PBS. The flushed and the non-flushed segments from the contralateral side were then fixed in 3% neutral buffered formalin and processed for routine histology. The numbers of spermatozoa in the flushings and in 40 histological sections from each segment were counted. Before ovulation, the majority of spermatozoa was detected in the vagina and the uterine segments, whereas after ovulation, significantly higher numbers of spermatozoa were present in the uterine tubal segments. The decreasing gradient in sperm numbers at 30 min and 3 h after mating between the vagina, the uterine segments, including the UTJ, and the uterine tubal segments indicated that the cervix and the UTJ served as barriers for sperm transport in the cat. The UTJ and the uterine crypts acted as sperm reservoirs before ovulation whereas the isthmus was a sperm reservoir around the time of ovulation. There was no difference in sperm numbers in the tissue sections between flushed and non-flushed segments, implying that the flushing technique only recovered some intraluminal spermatozoa while most of the spermatozoa remained in the epithelial crypts. This was further supported by the finding that significantly higher numbers of spermatozoa were recovered in the flushings at 30 min and 3 h after mating, when more spermatozoa were free in the lumina, than at 48 and 96 h after mating, when the majority of the spermatozoa were entrapped in the uterine epithelial crypts.     

Characterization of uterine leukocyte infiltration in gilts after artificial insemination

The objective of this study was to characterize the uterine leukocyte influx after artificial insemination (AI). After detection of oestrus with a boar at intervals of 1.5 h, seventy-two gilts were randomly assigned to a 2 x 3 x 4 factorial arrangement. AI was performed with 100 ml extended semen containing 5 x 10(9) spermatozoa (semen; n = 36) or 100 ml VSP semen extender (extender; n = 36) at one of three times after detection of oestrus: 12, 24 or 36 h (n = 24/time). The uterus was lavaged at 6, 12, 18 or 24 h (n = 18/time) after AI to determine the total number of uterine leukocytes. In addition, uterine lavage was performed on nine untreated gilts immediately after the detection of oestrus to establish a baseline number of leukocytes. The leukocyte response in all samples consisted predominately (92-99%) of polymorphonuclear neutrophilic granulocytes (PMNs). The mean number of PMNs recovered from the uteri of gilts treated with semen was greater than in gilts treated with extender and in untreated gilts (P < 0.01). The greatest number of PMNs in semen-treated gilts was found 12 h after AI (P < 0.01), and this number was sustained for 24 h. In contrast, the number of uterine PMNs recovered from extender-treated gilts reached a peak at 6 h and had declined by 12 h after AI (P < 0.05). It was concluded that an extensive influx of PMNs into the uterus is a normal sequence to AI. The consequences and importance of semen-induced uterine leukocytosis needs further investigation.     

Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas)

Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean ± SD) 73.0 ± 12.2, 38.4 ± 8.8, 88.0 ± 0.1, and 59.3 ± 15.7%, respectively. A series of GnRH injections (3 x 250 μg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 ± 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 ± 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 ± 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 ± 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 ± 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species.     

Side of gestation in dairy heifers affects subsequent sperm transport and pregnancy rates after deep insemination into one uterine horn

Effects of side of previous gestation on sperm transport and pregnancy rates after deep cornual insemination were evaluated in 1686 Friesian cows in their first lactational period. Only single ovulating animals were used. At insemination, semen was deposited deep into the uterine horn ipsilateral or contralateral to the preovulatory follicle. A total of 876 cows (52%) ovulated in the ovary ipsilateral to the postgravid horn, and 810 cows ovulated in the contralateral ovary. Semen was deposited into the previously nongravid uterine horn of 832 cows, and into the gravid horn of 854 cows. The pregnancy rate was higher (P < 0.00001) for semen deposition into the previously nongravid horn (46.6%) than for semen deposition into the gravid horn (35.7%). For inseminations ipsilateral to the side of impending ovulation, pregnancy rates were higher (P = 0.0004) when ovulations occurred on the opposite side to the postgravid horn than on the same side. Pregnancy rates were higher (P = 0.002) for contralateral inseminations when ovulations occurred on the same side to the postgravid horn than on the opposite side; they were higher (P = 0.0001) for total ipsilateral than for total contralateral inseminations. There was no difference between ipsilateral and contralateral inseminations (P = 0.64) when ovulation occurred ipsilateral to the postgravid horn, but pregnancy rates were higher (P < 0.00001) when ipsilateral insemination was carried out into the nonpostgravid horn. Results indicate that the side of gestation in dairy heifers affects subsequent pregnancy rates after deep insemination into one uterine horn, possibly by affecting sperm transport.     

Effect of time of artificial insemination on embryo sex ratio in dairy cattle

The objective of the present study was to examine whether different intervals between insemination and ovulation have an influence on the sex of seven-day-old embryos in dairy cattle. Cows were inseminated once with semen of one of two bulls of proven fertility between 36 h before ovulation and 12 h after ovulation. Time of ovulation was assessed by ultrasound at 4-h intervals. In total, 64 embryos were determined to be male or female. Of these 64 embryos, 51.6% were female. The sex ratio in the various insemination-ovulation intervals (early: between 36 and 20 h before ovulation; intermediate: between 20 and 8 h before ovulation; late: between 8 h before and 12 h after ovulation) did not significantly differ from the expected 1:1 sex ratio (50, 50 and 55% females, respectively). Bull (Bull A and B) and Parity (primiparous and multiparous) had no influence on the expected 1:1 sex ratio either. The number of cell cycles was similar for male and female (P = 0.23) embryos when quality of the embryo (P < 0.0001) was included in the model. The results of this study indicate that, in cattle, the interval between insemination and ovulation does not influence the sex ratio of seven-day-old embryos.     

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.     

Inducing ovulation and artificial insemination in the European brown hare (Lepus europaeus Pallas, 1778)

The aim of the study was to show whether it is possible to induce ovulation in the hare by GnRH analogue administration and to carry out an effective artificial insemination (AI). The research was carried out during the breeding and non-breeding season. During the breeding season, plasma progesterone concentrations increased on the 4th day after intramuscular injection of GnRH analogue (buserelin), indicating induced ovulation and corpus luteum development. Prostaglandin F(2)alpha (dinoprost) was an effective luteolytic agent on day 9. During the non-breeding season, the GnRH analogue injection does not cause an increase of progesterone. The 17beta-estradiol concentrations during the breeding and non-breeding season were similar. It was shown that after GnRH analogue administration and artificial insemination with semen diluted in Tris buffer extender 80% females delivered live young (39-43 days after artificial insemination), which proves the effectiveness of inducing ovulation in the hare by means of hormonal stimulation.     

Semen collection,cryopreservation and artificial insemination in the dromedary camel

Collection of semen with a bovine artificial vagina (AV) was attempted with each of 14 camels over a period of 2 years. Semen samples were evaluated, extended and cryopreserved. Frozen thawed semen, diluted cooled semen or whole semen was used to inseminate some female camels which were induced to ovulate with hCG. Males ejaculated semen into the AV in 74.6% collection attempts. The male copulated for at least 200s in 62.9% attempts. The remaining copulations were of shorter duration. Similarly, 49.3% ejaculates were at least 3ml of semen. Libido and donation of semen improved from December onwards and reached a peak after mid January with peak performance persisting until April. It declined during May. The majority of camels had lost libido and refuse to donate semen by the end of May. Camel semen is in gel form. While 35.9% of 203 semen samples exhibited no individual sperm motility, 28.5% exhibited low to fair grade individual sperm motility and only 35.4% exhibited >50% sperm motility. Differences existed between animals (P<0.01) and months (P<0.05) of collection, while effect of copulation time was not significant. Mass motility was not observed in camel semen. Individual sperm motility develops after liquefaction of semen. Addition of caffeine but not chymotrypsin improved the individual motility. The mean live percent sperm count and normal acrosome were 73.3+/-1.0 and 92.0+/-0.5, respectively. Only 51.1% of 45 semen samples with pre-freeze motility of >50% and 25% of 16 semen samples from low pre-freeze motility group with an overall success of 44.2% of 61 semen samples were successfully preserved. Wide variation was observed in the freezability of semen from different males. Attempts to impregnate female camels with liquid semen, frozen thawed semen and whole semen after hCG induced ovulation resulted in 0/10, 1/13 and 4/10 pregnancies.     

A review on reproduction in South American camelids

In this paper, aspects of reproductive physiology and endocrinology, as well as sexual behaviour in South American camelids are reviewed. Because of the many unique features of reproduction in these animals, the application of advanced breeding techniques that are routinely used in other domestic species has been slow and, in some cases, are not applicable. Relatively high embryonic loss and the capacity to carry only one offspring at a time limit production in females. Furthermore, some 20% of females do not conceive following mating. Research is needed to elucidate the causes of embryonic loss, particularly in relation to the preferential location of embryos in the left uterine horn and the apparent differential luteolytic activity of the two uterine horns. A fuller understanding of the endocrine changes and mechanisms accompanying folliculogenesis, estrus, induction of ovulation and luteal regression may led to treatments that provide better control of ovulation and enhance the quality and viability of eggs shed. In the male, the hormonal interactions involved in facilitating libido need to be established and the underlying causes of declining libido resulting from continued exposure to estrous females, identified. More importantly, there is a need to develop a routine method to collect semen from animals on farm, or in studs. Deficiencies in this area have long hampered the evaluation of sires through proper assessment of their semen quality and in the application of artificial insemination. In addition, establishment of techniques to freeze semen from these species has obvious advantages for breeding, including crossbreeding between species to improve products such as fibre and to assist in preservation of some of the more endangered New World camelids.     

Fertility after deep intra-uterine artificial insemination of concentrated low-volume boar semen doses

Boar semen can be successfully frozen - highly packed - in small containers (medium-straw, MS or MiniFlatPack, MFP). The use of deep intra-uterine artificial insemination (DIU-AI) can make possible the deposition of small volumes of this thawed, non re-extended semen deeply intra-uterine, close to the sperm reservoir. The present experiments studied the fertility achieved after single or double DIU-AI per oestrus, with special attention to the interval between AI and spontaneous ovulation. Semen from two boars of proven fertility was frozen in MS or MFP holding 1 x 10(9) total spermatozoa. Multiparous (2-5 parity, n=42) crossbred sows were checked for oestrous behaviour after weaning and the occurrence of spontaneous ovulation was checked with transrectal ultrasonography (TUS) to establish the mean interval between onset of oestrus (OO) and ovulation which was found to be when approximately 2/3 of the oestrus period has passed. The sows were, in the following standing oestrus, subjected to DIU-AI using thawed semen from either MS (n=20) or MFP (n=22), inseminated without further re-extension. The sows were randomly allotted to one of three groups: (1) single DIU-AI 8 h before expected ovulation (control group, n=19); (2) single DIU-AI 4 h before expected ovulation (treatment group S, n=15); and (3) double DIU-AI 12 and 4 h before expected ovulation (treatment group D, n=8). Occurrence of spontaneous ovulation was confirmed by TUS, performed as during the first oestrous period and used to determine the real interval of DIU-AI and ovulation. Pregnancy was also confirmed by TUS 28 days after OO in those sows not returning to oestrus. These sows were slaughtered (30-45 days of pregnancy), and the appearance of the reproductive tract and ovaries, the number of live and dead foetuses, of implantation sites and of corpora lutea (CL) were recorded. Sows (n=9) returning to oestrus ("open") were re-inseminated (either once [n=4] or twice [n=5]) the following oestrus with either MFP (n=5) or MS (n=4) and slaughtered 12-14 h post-ovulation for recovery of tubal oocytes and of spermatozoa from the uterotubal junctions (sperm reservoir), to assess the degree of effectiveness of sperm transport. Post-thaw sperm motility was 44.3+/-3.21% in MFP and 42.8+/-0.72% for MS (LSmean+/-S.E.M., n.s.), and did not significantly change from thawing to AI. The DIU-AI could be performed in all sows, but insertion was difficult (slow >5 min) in 5/42 sows. Four of these sows returned to oestrus. Pregnancy rate averaged 35% (group D: 25%, group S: 40%, control: 36%, n.s.). The interval between DIU-AIs and spontaneous ovulation varied largely, ranging from -13 to -3 h for group C, for group S from -11 to +3 h and for group D from -17 to -4 h. Pregnancy rates were clearly related to the interval DIU-AI and ovulation, being highest (60%, 12/20) when AI occurred between 8 and 4 h before spontaneous (not expected) ovulation. The number of implantation sites ranged 6-22 (n.s. among groups), and the number of alive foetuses 2-11 (n.s. among groups). Implantation rate (total number of implantations/CL) ranged 48.0-69.7% being highest in the D-group (P<0.05). The examination of the "open" sows slaughtered 12-14 h post-ovulation revealed few recovered oocytes were fertilized (approximately 10%). Only 40% of oocytes had spermatozoa bound to the zona pellucida, not more than two spermatozoa per oocyte. Moreover, low sperm numbers (approximately 4000) were found in the sperm reservoirs (UTJs), irrespective of using single or double DIU-AI (n.s.). The highest values (P<0.05) for these variables were recorded when DIU-AI (either single or double [second AI]) occurred 4-8 h before ovulation, especially when MFP-semen was used (P<0.05). In conclusion: (1) DIU-AI can be easily performed in most sows; (2) pregnancies can be obtained by the DIU-AI of low volumes of highly concentrated frozen-thawed boar semen, once or twice during oestrus, but fertility is still low, probably owing to an unsatisfactory sperm transport when expected and real ovulation differ; and (3) fertility is related to the interval DIU-AI and ovulation which should be -8 to -4 h of spontaneous ovulation and to the package, MFP having shown better results in vivo. The results stress the need for careful, and frequent, control of oestrus signs.     

The two to four-cell embryos as source tissue of the tetrapeptide preventing ovulations in the hamster.

A tetrapeptide compound, derived from oviductal contents at 40 hours post-coitus, prevents ovulation when injected subcutaneously into cyclic hamsters (Kent, '73). The present experiments were designed to determine whether the source of the tetrapeptide is from the ova, embryos, oviducts or semen. Oviducts were either untouched or ligated at the ovarian or uterine end to limit contents to embryos, semen or ova and the contents then recovered at 40 hours pc and injected into cyclic hamsters. The results indicate that the source of the tetrapeptide is the embryos.     

Ovulating induction methods in rabbit does: the pituitary and ovarian responses

The aim of this study was to compare the pituitary and ovarian responses in rabbit does subjected to different methods of ovulation induction. Forty-eight receptive females were randomly distributed into six groups (N = 8) and were inseminated with standard glass catheters. Buserelin intramuscular (BM) does were inseminated using a pool of fresh heterospermic semen and an intramuscular injection of 1 μg of buserelin acetate to induce ovulation. Buserelin intravaginal (BV) does were inseminated in a similar way, but ovulation was induced with the GnRH analogue (10 μg of buserelin acetate) combined with 0.5 mL of semen extender. The raw semen (R) and saline groups (S) were inseminated with undiluted semen or saline, respectively, without any inducer of ovulation. Another group (A) received lumbar anaesthesia (1.5 mL of 2% lidocaine), and only the empty catheter was introduced into the vagina. The AR does were treated the same way as group A but were inseminated with raw semen instead of an empty catheter. Blood samples were collected to determine the LH concentrations before and after AI (30, 60, 90, and 120 minutes). Ovulation, pregnancy, and conception rates were determined after euthanasia on day 14 post AI. Ovulating does had higher mean LH concentrations than nonovulating does (197.9 vs. 45.9 ng/mL; P < 0.05). The ovulation rates of buserelin intramuscular and intravaginal does were 100%, and the pregnancy rates were 87.5% and 100%, respectively. Rabbit does in groups A and AR did not ovulate and had similar mean plasma LH concentrations after 60 minutes compared with the S group (49.4 and 49.2 ng/mL vs. 41.6 ng/mL, respectively), which reached ovulation and pregnancy rates of 37.5%. Does inseminated only with raw semen had an ovulation rate of 75% and a pregnancy rate of 62.5%; they also demonstrated higher plasma LH concentrations than does of the S, A, and AR groups. In conclusion, ovulation in rabbit does can be induced by exogenous GnRH administration (im and intravaginal). The high plasma LH concentration and ovulation rate in the R group with respect to the S and A groups could weakly indicate the presence of some molecules in the seminal plasma that could act on or be absorbed by vaginal mucosa. Sensory stimulation and “seminal factors” probably exert a synergy on the ovulation response as demonstrated by the comparison of LH release and the ovulation response in the R, S, RA, and A groups.     

Effect of insemination time of frozen semen on incidence of uterine fluid in mares

Ninety five mares were inseminated with frozen semen either within 12 h before ovulation or within 8 h after ovulation. The effect of preovulatory versus postovulatory insemination (AI) on the subsequent detection of uterine fluid was studied. The overall pregnancy rate was 43% and this was not significantly influenced by preovulatory or postovulatory insemination. When mares were first examined 12 h after AI, 18 of 52 mares (35%) had accumulated uterine fluid. However, when mares were first examined 18 to 24 h after AI, only 6 of 43 mares (14%) had uterine fluid. Presence of intrauterine fluid significantly lowered pregnancy rates. Timing of insemination did not affect incidence of uterine fluid. Serum concentrations of estrogen and progesterone at time of insemination did not influence uterine clearance or pregnancy rates, but both hormones were higher at preovulatory than at postovulatory inseminations. We concluded that there was no evidence that postovulatory inseminations would predispose mares to persistence of uterine fluid after AI.     

Effect of timing of artificial insemination on gender ratio in beef cattle

It was recently reported that cows inseminated at approximately 10 or 20 h before an expected ovulation deliver predominately a bull or heifer calf, respectively. The objective of this study was to further investigate the effect of timing of insemination on the gender of offspring in cattle. Angus heifers (n = 41) and cows (n = 98) were used in the study. Heifers were synchronized with a 16-d treatment of melengestrol acetate followed 17 d later with an injection of PGF2alpha. Cows were synchronized with GnRH followed 7 d later with PGF2alpha. A HeatWatch electronic estrus detection system was used to determine the onset of estrus. Based on previous studies, it was assumed that ovulation occurs approximately 32 h after the onset of estrus. Therefore, animals were artificially inseminated at either 8 to 10 h (early; > or = 20 h before expected ovulation) or 20 to 25 h (late; < or = 10 h before expected ovulation) after the onset of estrus. Sixty to 80 d after insemination, ultrasonography was used to confirm pregnancy status and to determine the gender of fetuses. Gender of calves was subsequently confirmed at calving. Data were analyzed for effects of time of insemination and sire or semen batch on gender ratio, as well as any effect of length and/or intensity of estrus on conception rate and gender ratio. Twenty-nine of 41 heifers and 69 of 98 cows were detected in estrus after synchronization and were inseminated; 20 of 29 heifers and 48 of 69 cows were subsequently confirmed pregnant. Neither the length of estrus nor its intensity (number of mounts) had an effect on pregnancy rate or gender ratio (P > or = 0.418). Timing of insemination (early versus late) had no effect on gender ratio (P = 0.887). Semen from 13 sires representing 17 lots was used to inseminate the cows and heifers. No differences (P = 0.494) were detected in the gender ratios resulting from different sires or semen batches. In contrast to previous findings, our results indicate that inseminating beef cattle at approximately 20 or 10 h before an expected ovulation does not alter the gender ratio of the resultant calves.     

T-cell distribution in two different segments of the equine endometrium 6 and 48 hours after insemination

The T-cell response after the introduction of semen into the uterine cavity in the mare was studied by examining, immunohistochemically, the distribution of helper T-cells (CD4+) and cytotoxic T-cells (CD8+) in endometrial biopsy specimens. Endometrial tissue samples were obtained from twenty-five gynecologically healthy mares during estrus before and 6 or 48 h after deposition of a single dose of stallion semen. An increase (P=0.04) in the number of helper T-cells (CD4+) compared to pre-insemination values was observed in the uterine body in both groups, 6 and 48 h, after insemination. No significant variations in numbers of CD8+ cells were recorded either 6 or 48 h after insemination. There seems to be an early (6 h) recruitment of helper T-cells to the equine endometrium after semen deposition, which might be related to the activation of the endometritis-like reaction seen as part of the equine uterine immune defense during estrus.     

Sex ratio in rabbits following modified artificial insemination

The possibility of modifying the sex ratio of rabbit litters was examined in two experiments involving artificial insemination (AI) with fresh semen. Three time periods of AI, relative to ovulation, were used in Experiment 1: (a) control, GnRH was administered immediately after AI with ovulation estimated to occur 10-12h after AI; (b) early AI, GnRH was given 6h after AI so that ovulation was delayed until 16-18 h after AI; (c) late AI, GnRH was administered 6h before AI, which was performed 4-6h before ovulation. There were 13 does per treatment, and each doe was used in the same treatment for three AIs at 42-day intervals. The second experiment involved two treatments in which the does were inseminated as for the control in Experiment 1 and AI was performed using semen prepared in the normal manner (Treatment 1) or after centrifugation through 11 discontinuous Percoll gradients (Treatment 2). There were 20 does per treatment, and each doe was used in the same treatment for three AIs at 42-day intervals. The proportion of female kits produced in Experiment 1 was: control 41.7+/-19.1%, early AI 49.8+/-17.8%, and late AI 41.4+/-16.4%. These proportions did not differ significantly between treatments or from the expected 50:50 sex ratio. Fertility was reduced by the early (60.0%) and late (73.7%) AI treatments relative to control AI (80.0%), and the difference between early and control AI almost achieved statistical significance (P<0.07). In Experiment 2, the proportion of female kits was not affected by treatment (control, 51.1%; Percoll, 54.8%), and there was a similar level of fertility for both treatments (control, 76.0%; Percoll, 74.1%). Prolificacy and perinatal mortality were not affected by treatment in either experiment. It was concluded that neither the timing of insemination nor Percoll centrifugation of semen affected the sex ratio at birth of rabbit litters.