Reproductive status of endemic felid species in Latin American zoos and implications for ex situ conservation

Reproductive evaluations were conducted on 185 male cats representing eight endemic Latin American species that were maintained in 44 zoos and private facilities in 12 Latin American countries. Reproductive assessments (testicular measures, ejaculate quality, and blood testosterone/cortisol concentration) were used to establish normative values for large‐ and small‐sized cats in Latin American collections. Data also were analyzed using multiple regression to study the impact of proven breeder status, diet, and various animal housing combinations. Most felids (>95%) in the survey were of wild‐born origin, and <20% had produced offspring in captivity. Larger felids had bigger testes and produced more semen, but tended to produce low‐sperm‐density ejaculates. The ejaculates of small felids were more sperm‐concentrated, but contained fewer total spermatozoa. Sperm motility was unrelated to species size, and certain species (puma, margay, tigrina, and jaguarundi) consistently produced few (<40%) normal sperm forms. Across species, >50% of males had low sperm counts (<1 million total sperm per ejaculate). Among large cats (jaguars and pumas), proven breeders had larger (P<0.05) testes, greater semen volume, and more normal sperm than nonbreeders. Males on adequate diets had higher (P<0.05) circulating cortisol. Among small‐sized felids, proven breeders had higher (P<0.05) testosterone, and males housed alone or paired with a conspecific female had more (P<0.05) total sperm per ejaculate and greater (P<0.05) seminal and testicular volumes. Fifty‐nine ejaculates (potentially representing ～100 artificial insemination (AI) or 26,000 in vitro fertilization (IVF) procedures) were cryopreserved for a felid genome resource bank. In conclusion, breeding success and reproductive traits for many endemic felids in Latin American zoos appear to be suboptimal, and likely would benefit from improvements in diet and exhibitry. Technology transfer and continued training of zoo staff and scientists in Latin American countries are essential if these zoos are to achieve their tremendous conservation potential for felids and other threatened endemic species. Zoo Biol 22:421–441, 2003. © 2003 Wiley‐Liss, Inc.     

Induction of ovulation and successful artificial insemination in a Persian leopard (Panthera pardus saxicolor)

Techniques of artificial insemination have not been readily applied to zoo animals. The nonsurgical approach has been unsuccessful in nondomestic felids until the present study where pregnancy was achieved in a Persian leopard. The demonstration of this technique should encourage renewed attempts to artificially inseminate nondomestic felids.     

Sperm morphology is better in the second ejaculate than in the first in domestic cats electroejaculated twice during the same period of anesthesia

Several species produce ejaculates of inferior quality after a period of sexual abstinence, but the frequency of semen collection has thus far not been shown to affect sperm morphology in felids. The aim of this study was to determine whether sperm morphology and motility would differ between 2 ejaculates collected from the same cat within a short interval. Fifteen male domestic cats were anesthetized and then electroejaculated twice, with a 5- to 10-min interval between treatments. A standardized electroejaculation regimen was used with 80 stimuli, from 2 to 5 V, for each ejaculate. The first ejaculates contained significantly higher (P < 0.05) proportions of distal droplets, coiled tails and immotile spermatozoa than the second ejaculates, which contained significantly higher proportions of morphologically normal spermatozoa (40.9 vs 54.6%) but a lower sperm count (39.0 x 10(6) vs 5.2 x 10(6)). The higher proportions of defective spermatozoa and the lower motility in the first ejaculate than in the second were probably due to the aging of spermatozoa in the epididymis. These results show that the second ejaculate collected within a short interval has better sperm morphology and motility than the first and that this should be considered when evaluating semen quality in the domestic cat and when collecting cat semen to be used for artificial insemination or to be frozen for storage.     

New developments in low-dose insemination technology

New nonsurgical procedures for inseminating swine with a low number of spermatozoa have been developed and/or evaluated over the last few years. These procedures allow the deposition of the insemination dose into the uterine body (post-cervical insemination) or directly into the uterine horn (deep intrauterine insemination). With the use of the post-cervical insemination, a threefold reduction in the number of fresh sperm has been successfully used to achieve pregnancy. Using deep intrauterine insemination (DUI), up to a 20-fold reduction in the number of fresh spermatozoa or a sixfold reduction in the number of frozen/thawed spermatozoa can be achieved, with reproductive performance very similar to that obtained after standard AI. Complementing these nonsurgical insemination techniques, a new procedure for depositing spermatozoa into the oviduct by laparoscopy has been recently described. This laparoscopic technique has proven to be applicable to diluted and sex-sorted spermatozoa. The development of new insemination procedures will help achieve more efficient application of currently available sperm technologies. Using appropriate insemination procedures, it is now feasible to achieve high fertility rates with cooled, frozen-thawed, or sex-sorted semen.     

Early pregnancy loss in sows after low dose, deep uterine artificial insemination with sex-sorted, frozen-thawed sperm

Recent developments in reproductive technologies have enabled the production of piglets of a predetermined sex via non-surgical, low dose artificial insemination. The practical application of sex-sorting technology to the pig is made challenging by the large numbers of sperm required for successful insemination of sows. One way of overcoming the time required for sex-sorting may be to create a bank of cryopreserved, sex-sorted sperm, thus making available appropriate doses as sows require insemination. To date, little success has been achieved with non-surgical inseminations of sex-sorted boar sperm. This study attempted to achieve litters of a predetermined sex after a double insemination of sows with 160x10(6) sex-sorted, frozen-thawed sperm. Sows were synchronised and sperm were non-surgically inseminated into the proximal third of the uterine horn at 36 and 42 h after hCG administration. Sows inseminated with sex-sorted sperm achieved similar pregnancy rates to those receiving an equal dose of unsorted, frozen-thawed sperm. However, all sows conceiving after insemination with sex-sorted sperm returned to oestrus within 57 days of insemination. This was a higher rate of pregnancy loss than observed for sows inseminated with unsorted sperm (37.5%; P=0.031). A combination of low sperm numbers and potentially compromised developmental capability of embryos derived from sex-sorted sperm may have resulted in this early stage loss of pregnancy.     

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.     

Assessment of a new utero-tubal junction insemination device in dairy cattle

A new artificial insemination device for semen deposition near the utero-tubal junction in cattle (Ghent device) has been developed at the Ghent University (Belgium). In this study, the effect of the new insemination device on sperm quality was evaluated. Moreover, in a field trial 4064 dairy cows were inseminated by 12 inseminators to examine the efficacy of the device under field conditions.The Ghent device is a disposable plastic catheter which can easily follow the curvature of the uterine horns and thus reach the utero-tubal junction (UTJ). After expulsion of the inseminate with 0.7 or 1.7 ml of air, 19.0% of the insemination dose remained in the insemination catheter. Sperm loss can be diminished to 9.0% of the original insemination dose when the insemination catheter is flushed with 0.1 ml of air, followed by 0.6 ml of physiological saline solution. No toxic effect of the insemination catheter on sperm quality or fertilizing capacity was found. In the field trial, sperm were inseminated in dairy cattle which were divided in three groups. The first group was inseminated in the uterine body with the conventional insemination device, the second group in the uterine body with the Ghent device, and the third group in the tip of both uterine horns with the Ghent device. Each insemination was performed with 10 x 10(6) to 15 x 10(6) frozen-thawed spermatozoa. The pregnancy rates (PRs) were significantly affected by the insemination technique (P = 0.02), by the inseminator (P = 0.01), by heifer or cow (P < 0.01), and by the insemination number (P < 0.01). Pregnancy rates obtained with the conventional insemination device (57.6%) were significantly better than those obtained with the Ghent device in the uterine body (52.7%) (P < 0.01), but did not differ significantly from those obtained after deep insemination into both uterine horns (53.8%) (P = 0.27). It can be concluded that the Ghent device is suitable for utero-tubal junction insemination of dairy cattle under field conditions. Whether the Ghent device is also suitable for insemination with lower insemination doses is at present under investigation.     

Advances in deep uterine insemination: a fruitful way forward to exploit new sperm technologies in cattle

After clarifying regions of the female tract wherein spermatozoa are stored and the egg is fertilised, proposals are made for a modified site of sperm deposition in cattle. A deep pre-ovulatory insemination into the ipsilateral horn of the uterus-the side of the ovulatory follicle-should improve establishment of viable spermatozoa in the caudal region of the oviduct isthmus, the so-called functional sperm reservoir. Suppressed motility within viscous secretions and binding of sperm heads to endosalpingeal microvilli are features of this phase of storage. Activation and release of such spermatozoa would be prompted by imminent ovulation and associated ovarian endocrine programming by both local and systemic routes. Potential advantages of deep insemination include: (1) raising the fertility of genetically valuable bulls whose non-return rates are sub-optimal; (2) reducing the number of spermatozoa in each insemination dose; (3) exploiting the limited numbers of sex-selected sperm cells (X- and Y-chromosome-bearing spermatozoa) available from flow cytometry; (4) breeding from valuable but oligospermic bulls. Putative disadvantages might include: (1) rectal palpation of the ovaries to identify the pre-ovulatory follicle; (2) damage to or even perforation of the uterine wall by the insemination device; (3) the risk of polyspermic fertilisation; (4) specific training in the technique for non-clinically qualified inseminators. Each of these reservations receives comment. In conclusion, a modified technique of insemination should be feasible under commercial conditions, could be coupled with new sperm technologies, and would give a boost to the artificial insemination industry.     

Artificial insemination in Callithrix jacchus using fresh or cryopreserved sperm

Assisted reproductive techniques are needed urgently to facilitate the captive breeding of many New World primate species which are endangered in the wild and to assist the effective genetic management of small colonies. A protocol was devised for artificial insemination in the common marmoset, Callithrix jacchus, using ejaculated sperm obtained by vaginal washing after copulation. A double insemination protocol was employed, with the first insemination taking place the day before ovulation was expected to occur and the second 48 h later. All six females inseminated with fresh ejaculated sperm became pregnant, delivering a total of 16 offspring at term. The gestation lengths and litter sizes were not statistically different from those observed in pregnancies following natural mating. The insemination protocol was adapted for use with cryopreserved ejaculated sperm by including an additional insemination on the day of expected ovulation, to take into account differences in the capacitation time of frozen–thawed sperm compared to fresh sperm. Three out of six females inseminated according to this triple insemination schedule, conceived, although one female subsequently resorbed twin foetuses approximately 100 days later. The remaining two pregnant females delivered four babies at term, one singleton and one set of triplets. In the final group, six females were inseminated with low doses of cryopreserved epididymal sperm using the same triple insemination protocol used for frozen–thawed ejaculated sperm. One female conceived, delivering triplets.     

Assessment of human sperm function after recovery from the female reproductive tract

The physiology and fertile life of human spermatozoa in the female reproductive tract have received little previous attention. A technique was developed for recovering spermatozoa from human cervical mucus at various intervals after artificial insemination. The functions of these cells as measured by penetration of the human zona pellucida and fusion with the zona-free hamster oocytes were examined. Penetration into the zona pellucida was consistently observed when sperm were recovered from 1 to 80 h after insemination. Penetration through the zona into the perivitelline space (PVS) was seen from 1 to 72 h after insemination. Fusion of human sperm with zona-free hamster oocytes was observed from 1 to 48 h after insemination. Motile sperm were recovered 112 and 120 h after insemination with swimming speeds comparable to freshly capacitated spermatozoa. Concentrations of recovered sperm at these longer intervals from insemination were insufficient for sperm-oocyte assays. These studies demonstrate that human spermatozoa aged in vivo may be recovered from cervical mucus for physiologic study, and suggest that the fertile life of human sperm may be 80 h or more.     

Low dose insemination in cattle with the Ghent device

A new artificial insemination device for semen deposition near the uterotubal junction (UTJ) in cattle (Ghent device) was developed at Ghent University (Belgium). In this study, UTJ insemination of dairy cows with the Ghent device was compared with the conventional insemination technique to evaluate the effect on pregnancy rates after insemination with different doses of semen. In each of three field trials, the cows (n=795, 659, 360) and heifers (n=253, 182, 231) were randomly assigned to receive 12 million sperm deposited in the uterine body using conventional techniques (control) or a reduced sperm dose (RSD) deposited in the same manner as the control or bilateral deposition near the uterotubal junction using the Ghent device (Ghent). Sperm dosages for RSD and Ghent inseminations were 8, 4, and 2 million sperm for field trials 1-3, respectively. In the multivariable analysis, the pregnancy rates were significantly affected by the parity of the cow (p相似文献   

Preselection of sex of offspring in swine for production: current status of the process and its application

It is estimated that as many as 30,000 offspring, mostly cattle, have been produced in the past 5 years using AI or some other means of transport with spermatozoa sexed by flow cytometric sperm sorting and DNA as the marker of differentiation. It is well documented that the only marker in sperm that can be effectively used for the separation of X- and Y-chromosome bearing spermatozoa is DNA. The method, as it is currently used worldwide, is commonly known as the Beltsville Sperm Sexing Technology. The method is based on the separation of sperm using flow cytometric sorting to sort fluorescently (Hoechst 33342) labeled sperm based on their relative content of DNA within each population of X- and Y-spermatozoa. Currently, sperm can be produced routinely at a rate of 15 million X- and an equal number of Y-sperm per hour. The technology is being applied in livestock, laboratory animals, and zoo animals; and in humans with a success rate of 90-95% in shifting the sex ratio of offspring. Delivery of sexed sperm to the site of fertilization varies with species. Conventional AI, intrauterine insemination, intra-tubal insemination, IVF with embryo transfer and deep intrauterine insemination are effectively used to obtain pregnancies dependent on species. Although sperm of all species can be sorted with high purity, achieving pregnancies with the low numbers of sperm needed for commercial application remains particularly elusive in swine. Deep intrauterine insemination with 50-100 million sexed boar sperm per AI has given encouragement to the view that insemination with one-fiftieth of the standard insemination number will be sufficient to achieve pregnancies with sexed sperm when specialized catheters are used. Catheter design, volume of inseminate, number of sexed sperm are areas where further development is needed before routine inseminations with sexed sperm can be conducted in swine. Cryopreservation of sex-sorted sperm has been routinely applied in cattle. Although piglets have been born from frozen sex-sorted boar sperm, freezing and processing protocols in combination with sex-sorted sperm are not yet optimal for routine use. This review will discuss the most recent results and advances in sex-sorting swine sperm with emphasis on what developments must take place for the sexing technology to be applied in commercial practice.     

Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency

Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X-bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.     

Timing of insemination using sex-sorted sperm in embryo production with Bos indicus and Bos taurus superovulated donors

Two experiments were designed to evaluate the effect of different insemination times (12 and 24h or 18 and 30h) and different types of semen (sex-sorted or non-sorted sperm) on embryo production in Nelore (Bos indicus) and Holstein (Bos taurus) superstimulated donors. In the first experiment, hormonal superstimulation of ovarian follicular development in Nelore donors (n=71) was performed in randomly allocated animals to one of the three treatment groups, and they were inseminated at 12 and 24h after an ovulatory stimulus with pLH treatment was applied, either with sex-sorted (4.2×10(6) sperm/insemination; S12/24; n=17) or non-sorted sperm (20×10(6) sperm/insemination; NS12/24; n=18), or they were inseminated at 18 and 30h using sex-sorted sperm (4.2×10(6) sperm/insemination; S18/30; n=19). A greater number of transferable embryos were found when sex-sorted sperm was used to inseminate the animals at 18 and 30h (4.5±3.0) compared to insemination at 12 and 24h (2.4±1.8; P<0.001). However, a greater embryo production (6.8±2.6) was obtained with non-sorted sperm. In the second experiment, the same insemination times and semen types were used in lactating high-production Holstein cows (n=12). A crossover design was employed in this trial. A lesser embryo production (P=0.007) was found in Holstein donors that were inseminated using sex-sorted sperm at 12 and 24h (4.6±3.0) compared to non-sorted sperm (8.7±2.8). However, intermediate results were obtained when the inseminations with sex-sorted sperm were performed at 18 and 30h (6.4±3.1). These results supported the current hypothesis that it is possible to improve embryo production using sex-sorted sperm in B. indicus and B. taurus superstimulated donors when the inseminations are performed near the same time as time-synchronized ovulations. However, the embryo production for timed artificial insemination (TAI) with sex-sorted sperm was still less than the production with non-sorted sperm.     

Sperm mortality, insemination and fertilization in the damselfly Ischnura senegalensis: comparisons between wild and inbred populations

Inbreeding can have deleterious effects on individual or population fitness. To avoid fitness reduction, individuals may adopt behavioral or physiological mechanisms to reduce their investment in the production of offspring with genetically similar mates. We examined whether insemination by inbred males introduced more dead sperm than insemination by wild males by counting sperm in female Ischnura senegalensis (Rambur) sperm storage organs. If inbred males inseminated fewer or lower-quality sperm, females would avoid inferior sperm. Our results revealed three features of damselfly inbreeding: insemination failed in a larger proportion of inbred pairs than in wild pairs, inbred pairs showed significantly reduced fertility, and the numbers of live and dead sperm in an inbred female’s sperm storage organs did not differ from those in wild females. These results suggested that neither sperm quantity nor sperm quality was responsible for low fertility to a significant extent, but some kind of female quality, such as sperm usage or storing ability, was. Although inbred pairs had lower fertility, there were no significant differences between inbred and wild pairs in the total numbers of live or dead sperm. It thus seemed that female choice at the insemination stage was responsible for low fertility rather than sperm quantity or quality measured by live-to-dead ratio.     

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.     

Electron microscopic observations on sperm entry and pronuclear formation in naked eggs of the rose bitterling in polyspermic fertilization

Unfertilized eggs of the rose bitterling (Rhodeus ocellatus ocellatus) were squeezed out of females that had an elongated ovipositor and were dechorionated mechanically with fine forceps in physiological saline. The dechorionated eggs were transferred into fresh water then inseminated at once by spermatozoa of the same species. A large number of spermatozoa was found on the surface of eggs that had not yet had cortical reaction following insemination. The surface of the naked eggs responded by formation of many small cytoplasmic protrusions (viz., fertilization cones) at sperm attachment sites. The formed fertilization cones were rosettelike structures formed by the aggregation of some bleblike swellings devoid of microvilli and microplicae. About 10 min after insemination, the fertilization cones retracted, but marks of their presence characterized by less microvilli and microplicae remained in the eggs 15 min after insemination. Many spermatozoa penetrated into the cytoplasm of each naked egg. The sperm nuclear envelope disappeared by means of vesiculation resulting from fusion of the inner and outer membranes. The sperm nucleus decondensed and developed into a larger male pronucleus. Smooth-surfaced vesicles surrounded the decondensing sperm nucleus and formed the new male pronuclear envelope. Sperm mitochondria and flagella were found in the egg 15 min after insemination. The response of the egg surface to sperm entry and pronucleus formation are discussed.     

Insemination factors related to timed AI in cattle

Six-day-old bovine ova/embryos were recovered non-surgically and used as biomonitors to evaluate time of artificial insemination. These embryos/ova provided information regarding fertilization status and embryo quality, as well as quantitative and qualitative data regarding associated accessory sperm. Both sperm access to the ovum (addressed by accessory sperm) and fertilization status/embryo quality were important in addressing pregnancy rate for specific intervals from the onset of estrus to insemination. Based on these biomonitors, early insemination failed to achieve optimum pregnancy rate due to inadequate access of sperm to the ovum (i.e., low fertilization rate, manifested by low accessory sperm numbers). However, embryo quality was high in early inseminations, which favors pregnancy. Late insemination failed to achieve optimum pregnancy rate (due to reduced embryo quality), however, sperm access to the ovum was highest. Thus, the selection of an insemination time to achieve optimum pregnancy rate appeared to be a compromise between the two extreme intervals. For timed-AI programs, consideration of the time of ovulation (and its variability) becomes important, in addition to conventional considerations, such as semen handling, site of insemination, and bull selection.     

Pregnancy rates after artificial insemination with cooled stallion spermatozoa either with or without Single Layer Centrifugation

A successful outcome after artificial insemination with cooled semen is dependent on many factors, the sperm quality of the ejaculate being one. Previous studies have shown that spermatozoa with good motility, normal morphology, and good chromatin integrity can be selected by means of colloid centrifugation, particularly single layer centrifugation (SLC) using species-specific colloids. The purpose of the present study was to conduct an insemination trial with spermatozoa from “normal” ejaculates, i.e., from stallions with no known fertility problem, to determine whether the improvements in sperm quality seen in SLC-selected sperm samples compared with uncentrifuged controls in laboratory tests are reflected in an increased pregnancy rate after artificial insemination. In a multicentre study, SLC-selected sperm samples and uncentrifuged controls from eight stallions were inseminated into approximately 10 mares per treatment per stallion. Ultrasound examination was carried out approximately 16 days after insemination to detect an embryonic vesicle. The pregnancy rates per cycle were 45% for controls and 69% for SLC-selected sperm samples, which is statistically significant (P < 0.0018). Thus, the improvement in sperm quality reported previously for SLC-selected sperm samples is associated with an increase in pregnancy rate, even for ejaculates from stallions with no known fertility problem.