TRY-5 is a sperm-activating protease in Caenorhabditis elegans seminal fluid

Seminal fluid proteins have been shown to play important roles in male reproductive success, but the mechanisms for this regulation remain largely unknown. In Caenorhabditis elegans, sperm differentiate from immature spermatids into mature, motile spermatozoa during a process termed sperm activation. For C. elegans males, sperm activation occurs during insemination of the hermaphrodite and is thought to be mediated by seminal fluid, but the molecular nature of this activity has not been previously identified. Here we show that TRY-5 is a seminal fluid protease that is required in C. elegans for male-mediated sperm activation. We observed that TRY-5::GFP is expressed in the male somatic gonad and is transferred along with sperm to hermaphrodites during mating. In the absence of TRY-5, male seminal fluid loses its potency to transactivate hermaphrodite sperm. However, TRY-5 is not required for either hermaphrodite or male fertility, suggesting that hermaphrodite sperm are normally activated by a distinct hermaphrodite-specific activator to which male sperm are also competent to respond. Within males, TRY-5::GFP localization within the seminal vesicle is antagonized by the protease inhibitor SWM-1. Together, these data suggest that TRY-5 functions as an extracellular activator of C. elegans sperm. The presence of TRY-5 within the seminal fluid couples the timing of sperm activation to that of transfer of sperm into the hermaphrodite uterus, where motility must be rapidly acquired. Our results provide insight into how C. elegans has adopted sex-specific regulation of sperm motility to accommodate its male-hermaphrodite mode of reproduction.     

Early morphological events of in vitro fertilized bovine oocytes with frozen-thawed spermatozoa

Bovine follicular oocytes were matured and inseminated in vitro with spermatozoa capacitated in vitro. The first evidence of sperm penetration was observed at 3 h after insemination. The penetration rate increased until 5 h, and reached a maximum rate (92%) at 5 h. Decondensation of the sperm head and pronuclear formation were observed 4 h and 7 h after insemination, respectively. Female chromatins of all penetrated oocytes were activated at 3 h, and female pronuclei were formed at 7 h after insemination. Percentages of oocytes with male and female pronuclei at 9 h were 88 and 94%. Polyspermy (4, 7, 19 and 29% at 4, 5, 7 and 9 h after insemination, respectively) and abnormal development of male pronuclei (6 and 7% at 7 and 9 h after insemination, respectively) were also seen.     

GnRH-agonist induction of fertile estrus with either natural mating or artificial insemination, followed by birth of pups in gray wolves (Canis lupus)

Although captive populations of endangered species such as the Mexican gray wolf (Canis lupus baileyi) can benefit from artificial insemination to accomplish genetic exchange, reliable techniques for timing insemination are lacking. We used the generic gray wolf (C. lupus) to test the efficacy of a short-acting GnRH-agonist implant, deslorelin, for inducing estrus. Of five females receiving implants on 17 or 18 January 2003, two mated naturally 10-17 days later, and the others were artificially inseminated using fresh semen, one on day 7 and all three on day 11. Relaxin tests revealed that one artificially inseminated female and both naturally mated females were pregnant on 1 March, and all three gave birth to healthy puppies on 4-6 April. Of the artificially inseminated females, only the one who subsequently conceived and gave birth was judged to be in cytologic estrus at the time of insemination. Two females were treated again with deslorelin on 12 January 2004, followed by collection of fecal samples for hormone analysis. One female, who was housed with a male, copulated on day 17 but did not conceive; the other was not with an adult male. Fecal progestin and estrogen profiles suggested that estrus, but not ovulation, was induced. These results indicated that deslorelin could induce fertile estrus in the gray wolf, although individual response varied. Further investigation is needed to better define and control the interval between implant insertion and ovulation for optimal timing of insemination.     

Protamine dissociation before decondensation of sperm nuclei during in vitro fertilization of pig oocytes

The correlation between morphological changes and the dynamics of protamine in boar sperm chromatin during in vitro fertilization of pig oocytes matured in vitro was assessed. For this purpose, protamine was purified from boar sperm nuclei and an antiserum against protamine was developed. After affinity purification, the antiserum reacted exclusively with boar protamine during western blotting, showing no crossreactivity with core histones. Immunohistochemical evaluation revealed that only fully developed spermatid nuclei in boar testes stained strongly with the antiserum. When pig oocytes matured in vitro were fertilized in vitro, sperm penetration was observed in 37% of oocytes at 2 h after insemination and the penetration rate increased to 99% by 5 h after insemination, accompanied by an increase in polyspermic penetration. Paraffin wax sections of the inseminated oocytes were examined by immunohistochemical analysis with the antiserum. The proportion of condensed sperm nuclei that reacted with the antiserum was 87% of the sperm nuclei that penetrated by 2 h after insemination, and this decreased to 20 and 13% at 3 and 5 h after insemination, respectively. However, none of the decondensing sperm nuclei or male pronuclei reacted with the antiserum during the entire insemination period. These results indicate that a specific antiserum against boar protamine can be raised and, using this serum, it has been demonstrated that protamine is dissociated from boar sperm nuclei before decondensation during in vitro fertilization.     

Long-term sperm storage in the Siberian crane (<Emphasis Type="Italic">Grus leucogeranus</Emphasis> pallas): Analysis of paternity and relatedness under artificial insemination

Using ten microsatellite loci, paternity analysis has been conducted for 71 individuals of the Siberian crane (Grus leucogeranus Pallas) obtained under artificial insemination in Oka Crane Breeding Center in 2001–2014. The fathers of 39 chicks were the sires whose sperm was used for insemination directly before fertilized egg laying. Paternity of 23 fertilizations belonged to the sires whose sperm was used in the beginning or middle of insemination cycle. Nine cases of fertilization resulted from natural copulation of artificially inseminated females with their social partners. The terms of sperm storage in the female’s reproductive ducts before fertilization were 0–6 days in the case of paternity of the last sperm donor and 2–15 days in the case of competing sperm by previous donors. Genetic relatedness by microsatellite loci between breeders of the captive Siberian crane population does not prevent fertilization and does not always lead to inbreeding depression.     

Transplantation of male pronucleus derived from in vitro fertilization of enucleated oocyte into parthenogenetically activated oocyte results in live offspring in mouse

In this study, inter-strain reconstructed embryos were produced by combining the female pronucleus of Kunming mouse (white) with male pronucleus of C57BL/6 strain (black). Metaphase II (MII) oocytes of Kunming mouse were enucleated and the zona pellucida was removed. Then, the enucleated oocytes were inseminated by capacitated sperm of C57BL/6 mouse in vitro. At the same time, MII oocytes of Kunming mouse were artificially activated using strontium chloride solution, which did not contain cytochalasin B. Finally, we removed the male pronucleus derived from C57BL/6 sperm and injected it into a parthenogenetically activated one-pronucleus oocyte by micromanipulation. The reconstructed 2-cell embryos were transplanted into the oviducts of 22 foster mother mice, each receiving about 20 embryos. In the end, seven healthy and live pups were born from one recipient.     

Sperm influences female hibernation success, survival and fitness in the bumble-bee Bombus terrestris

We present evidence that in the absence of the transfer of male gland compounds in the ejaculate as well as of behavioural male traits, such as mate guarding or harming of females, sperm itself affects female life-history traits such as hibernation success, female longevity and female fitness. Using the bumble-bee Bombus terrestris, we artificially inseminated queens (females) with sperm from one or several males and show that sire groups (groups of brother males) vary in their effects on queen hibernation survival, longevity and fitness. In addition, multiply inseminated queens always had a lower performance as compared to singly inseminated queens. Apart from these main effects, sire groups (in situations of multiple insemination) affected queen longevity and fitness not independently of each other, i.e. certain sire group combinations were more harmful to queens than others. So far, the cause(s) of these effects remain(s) elusive. Harmful male traits as detected here are not necessarily expected to evolve in social insects because males depend on females for a successful completion of a colony cycle and thus have strong convergent interests with their mates.     

Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting

Intact, viable X and Y chromosome-bearing sperm populations of the rabbit were separated according to DNA content with a flow cytometer/cell sorter. Reanalysis for DNA of an aliquot from each sorted population showed purities of 86% for X-bearing sperm and 81% for Y-bearing sperm populations. Sorted sperm were surgically inseminated into the uterus of rabbits. From does inseminated with sorted X-bearing sperm, 94% of the offspring born were females. From does inseminated with sorted Y-bearing sperm from the same ejaculates, 81% of the offspring were males. The probability of the phenotypic sex ratios differing from 50:50 were p less than 0.0003 for X-sorted sperm and p less than 0.004 for Y-sorted sperm. Thus, the phenotypic sex ratio at birth was accurately predicted from the flow-cytometrically measured proportion of X- and Y-bearing sperm used for insemination.     

Penetration of mouse eggs at various intervals after insemination as influenced by concentration of sperm and strain of male

This study was conducted to determine the extent to which the percentage of mouse eggs that were penetrated by sperm at the end of the period of sperm penetration was due to the proportion of eggs penetrated per unit of time and to the span of time of sperm penetration. Female mice of ICR strain were inseminated 1.5 hr after ovulation with 5 X 10(6) sperm/50 microliter from males of DBA/2N, CF1 or C57BL/6N strains to determine the effect of the male. To determine the effect of concentration of sperm ICR females were inseminated with 2, 4, 6, or 8 X 10(6) sperm/50 microliter from CF1 males. Females were killed at various intervals after insemination and the eggs were recovered and examined for evidence of penetration by a sperm. The time intervals from both insemination to the onset of egg penetration and from insemination to cessation of penetration were similar for the three strains of males. Throughout the period of penetration of eggs a constant percentage of eggs was penetrated per hour for a particular strain of male. The relative percentage penetrated per hour very closely approximated the relative percentage of eggs finally penetrated for each strain of male. The percentage of eggs penetrated per hour was linearly positively related to the concentration of sperm inseminated. The final percentage of eggs penetrated depended primarily on the rate at which the eggs were penetrated during the period of sperm penetration and not on the length of the period of egg penetration which was constant.     

Effect of Different Sperm Numbers on Fertility after Artificial Insemination of Foxes

A total of 325 blue for vixens were inseminated with fresh semen from 50 silver fox males. Each ejaculutate was divided into 4 portions and diluted so as to contain 100, 60, 40, and 20 million sperm/ml. Vixens in groups 1,2,3 and 4 had been randomly assigned to their group at the time of insemination. The animals were inseminated once with either 100,60,40, or 20 million sperm. Vixens in groups 5 and 6 were selected by the technician after detecting signs of estrus during a physical examination. Animals judged to be at their optimal time for conception were assigned to group 5 and inseminated once with 20 million sperm. Animals considered to be early in their heat were assigned to group 6 and inseminated twice within 24 to 36 h with 20 million sperm per insemination dose. All inseminations were performed within 3 h of semen collection. A 1-ml total volume of extended semen was used for intrauterine deposition. In the random group inseminated once with 20 million sperm (group 4), both pregnancy rate and litter size were lower compared to the other random groups (groups 1,2, and 3), although the difference was not statistically significant. Among the vixens inseminated with 20 million sperm (group 4, 5, and 6) there was a significant difference in fertility between animals randomly selected and inseminaed once and those selected by the technician and inseminated twice (group 6). Our results suggest that for the crossbreeding of foxes 20 million sperm is the minimum insemination dose required for acceptable fertility with the present tecnique for sperm preservation and estrous determination.     

Effects of semen plasma from different fractions of individual ejaculates on IVF in pigs

We applied IVM/IVF techniques to investigate effects of preincubation of sperm with different fractions of semen plasma harvested from fresh ejaculates on in vitro penetration and fertilization of in vitro matured oocytes. Three fractions of semen plasma were separated from the complete ejaculate of three Landrace boars and used to coincubate sperm obtained from the first sperm-rich fraction of the same ejaculates. After 14 to 16 h coincubation at room temperature, sperm were preincubated in capacitation medium and then inseminated into fertilization medium containing porcine oocytes matured in vitro. The semen plasma used for coincubation affected penetration rate (P < 0.001); Sperm coincubated with Fraction 1 semen plasma had a higher penetration rate compared with sperm coincubated with Fraction 2 (P < 0.05), but not with Fraction 3. Boar affected male pronucleus formation rates after insemination (P < 0.05), but no difference among boars was found in monospermy rate, average number of sperm penetrating into each fertilized oocyte, or the average number of sperm attached. No boar by fraction interaction was found for any parameters studied.     

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.     

A quantitative comparison of the passage of capacitated and uncapacitated hamster spermatozoa through the uterotubal junction.

Female hamsters were artificially inseminated at the time of ovulation with an equal concentration and volume of capacitated sperm suspension in one uterus and uncapacitated sperm suspension in the contralateral uterus. When oviducts were examined 3.5-4.0 h after insemination, a significantly (paired t-test, p less than 0.05) lower number of spermatozoa were found in the oviduct from the side inseminated with capacitated sperm suspension compared to the side inseminated with uncapacitated sperm suspension. The reduction in the number of spermatozoa entering the oviduct on the side inseminated with capacitated sperm suspension was particularly evident when nearly all the spermatozoa in the suspension were hyperactivated. These results suggest that hamster spermatozoa require a progressive linear type of motility pattern to pass efficiently through the uterotubal junction and that under normal conditions in vivo, fertilizing spermatozoa initiate hyperactivated motility after entering the oviduct.     

Sperm precedence in the deer tick Ixodes dammini

ABSTRACT We determined whether female deer ticks Ixodes dammini Spielman, Clifford, Piesman & Corwin (Acari: Ixodidae) can be inseminated repeatedly and whether sperm from first or second matings take precedence in fertilizing eggs. Such information is essential to the design of attempts to reduce the fertility of these vectors of Lyme disease. Although spermatophores are present in about half of questing female ticks, they are present in virtually all those found on deer; the abundance of males on deer exceeds that of females and copulation is common. Females must be inseminated before commencing the rapid engorgement phase of feeding. Males need not be in attendance during feeding, provided that the female has been inseminated preprandially. Thus, preprandial insemination suffices to stimulate rapid engorgement, but less blood is taken than when the female is perprandially inseminated. Both types of insemination effectively fertilize eggs. Eggs from females sequentially inseminated by irradiated and non irradiated males, were fertilized mainly by sperm from the last male. Cobalt-irradiated males mate effectively and their sperm compete with those of non-irradiated males. Sperm from the second two sequential inseminations fertilize most of the eggs. By infesting deer with such irradiated male I.dammini , the abundance of these vector ticks may effectively be reduced.     

Development of human male pronucleus: Ultrastructure and timing

The process of human male pronuclear formation was studied using an experimental model based on in vitro inseminated human zona-free eggs prepared from oocytes that failed to fertilize in a clinical in vitro fertilization program. The main ultrastructural changes in penetrated sperm nuclei transforming into pronuclei were used to define four stages of pronuclear development. The first two stages, representing partial (Stage 1) and total (Stage 2) sperm chromatin decondensation, appeared as early as 1 hr after mixing of gametes. This rapid initial phase was followed by a more lengthy array of events leading to transformation of decondensed sperm nuclei into fully developed male pronuclei (Stages 3 and 4). Stage 3 was characterized by reformation of the nuclear envelope, reorganization of chromatin, and the assembly of nuclcolar precursors. It was not completed until 12 hr after in vitro insemination when fully developed male pronuclei (Stage 4) were first observed. In some eggs pronuclei did not reach Stage 4 at all. The results of this study provide a morphological background for further research into molecular aspects of human male pronuclear development and its regulation.     

High multiple paternity and low last-male sperm precedence in a hermaphroditic planarian flatworm: consequences for reciprocity patterns

It is difficult to predict a priori how mating success translates into fertilization success in simultaneous hermaphrodites with internal fertilization. Whereas insemination decisions will be determined by male interests, fertilization will depend on female interests, possibly leading to discrepancies between insemination and fertilization patterns. The planarian flatworm Schmidtea polychroa, a simultaneous hermaphrodite in which mating partners trade sperm was studied. Sperm can be stored for months yet individuals mate frequently. Using microsatellites, maternity and paternity data were obtained from 748 offspring produced in six groups of 10 individuals during four weeks. Adults produced young from four mates on average. Reciprocal fertilization between two mates was found in only 41 out of 110 registered mate combinations, which is clearly less than what is predicted from insemination patterns. Multiple paternity was high: > 80% of all cocoons had two to five fathers for only three to five offspring per cocoon. Because animals were collected from a natural population, 28% of all hatchlings were sired by unknown sperm donors in the field, despite a 10-day period of acclimatization and within-group mating. This percentage decreased only moderately throughout the experiment, showing that sperm can be stored and used for at least a month, despite frequent mating and sperm digestion. The immediate paternity a sperm donor could expect to obtain was only about 25%. Male reproductive success increased linearly with the number of female partners, providing support for Bateman's principle in hermaphrodites. Our results suggest that hermaphrodites do not trade fertilizations when trading sperm during insemination, lending support to the view that such conditional sperm exchange is driven by exchange of resources.     

Effect of follicle cells on in vitro fertilization of pig follicular oocytes

We investigated the effect of cumulus and granulosa cells (follicle cells) on in vitro fertilization of pig follicular oocytes matured in vitro. Oocytes surrounded by cumulus and connected with a piece of parietal granulosa cells (complexes) were matured in vitro for 46hours and were then divided into 4 groups: Group I oocytes were surrounded by expanded cumulus and granulosa cells; Group II oocytes were surrounded by expanded cumulus cells; Group III were denuded oocytes; and Group IV were denuded oocytes with cumulus cells from other complexes. After incubation for 4 hours and 40 minutes with frozen, thawed and preincubated pig epididymal spermatozoa, the oocytes were cultured for 5 hours and 20 minutes. When oocytes were inseminated in the presence of cumulus cells, the penetration rates were higher (92.5% for Group II and 89.5% for Group IV) than when cumulus cells were not used for insemination (Group III, 66.8%) or when oocytes with follicle cells were inseminated (Group I, 72.3%). Denudation of follicle cells before insemination (Group III) decreased the percentage of male pronuclear formation (50.8%) compared with that of oocytes surrounded by follicle cells (66.7% for Group II and 80.2% for Group I). These results support the ability of a moderate number of follicle cells to facilitate sperm penetration of pig follicular oocytes and male pronuclear formation.     

Production of transgenic pigs mediated by pseudotyped lentivirus and sperm

Sperm-mediated gene transfer can be a very efficient method to produce transgenic pigs, however, the results from different laboratories had not been widely repeated. Genomic integration of transgene by injection of pseudotyped lentivirus to the perivitelline space has been proved to be a reliable route to generate transgenic animals. To test whether transgene in the lentivirus can be delivered by sperm, we studied incubation of pseudotyped lentiviruses and sperm before insemination. After incubation with pig spermatozoa, 62±3 lentiviral particles were detected per 100 sperm cells using quantitative real-time RT-PCR. The association of lentivirus with sperm was further confirmed by electron microscopy. The sperm incubated with lentiviral particles were artificially inseminated into pigs. Of the 59 piglets born from inseminated 5 sows, 6 piglets (10.17%) carried the transgene based on the PCR identification. Foreign gene and EGFP was successfully detected in ear tissue biopsies from two PCR-positive pigs, revealed via in situ hybridization and immunohistochemistry. Offspring of one PCR-positive boar with normal sows showed PCR-positive. Two PCR-positive founders and offsprings of PCR-positive boar were further identified by Southern-blot analysis, out of which the two founders and two offsprings were positive in Southern blotting, strongly indicating integration of foreign gene into genome. The results indicate that incubation of sperm with pseudotyped lentiviruses can incorporated with sperm-mediated gene transfer to produce transgenic pigs with improved efficiency.     

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.     

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.