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Microfluidic technology can be used for sperm separation. Microfluidic devices generate a fluid flow to sort sperm from a media reservoir into a collection chamber. In the human and mouse, the use of microfluidic devices resulted in the selection of sperm with improved sperm motility, normal morphology, and DNA integrity for in vitro fertilization (IVF), intrauterine insemination (IUI), and intracytoplasmic sperm injection (ICSI). With the use of microfluidic sperm separation, centrifugation can be eliminated, diminishing the risk of reactive oxygen species exposure and DNA damage. We hypothesised that equine sperm can be separated using a microfluidic sorting device (Fertile PlusTM Sperm Sorting Chip; DxNow, Worcester, MA, USA) to improve the quality of sperm for ICSI. The aim of our research was to evaluate sperm parameters, including motility, morphology, membrane integrity, and DNA integrity, in frozen-thawed samples of equine semen before and after sorting using the Fertile Plus Sperm Sorting Chip. Two experiments were performed. In Experiment 1, the microfluidic device was used to separate frozen-thawed semen samples (n = 10) from research stallions (n = 3) with good quality frozen semen; all semen was frozen by one method in our laboratory. In Experiment 2, clinical samples of frozen-thawed semen (n = 11) from 7 stallions were evaluated. The semen was of variable quality and frozen at different facilities. Sperm analyses included (1) motility, (2) morphology (Hancock stain, Animal Reproduction Systems, Chino, CA, USA), (3) live-dead sperm (Hancock stain), (4) membrane integrity (HOS, hypo-osmotic swelling test), and (5) DNA fragmentation (SCD, sperm chromatin dispersion). Two sample t-tests were used to compare sperm parameters. In Experiment 1, use of the Fertile Plus Sperm Sorting Chip improved sperm parameters between the original and sorted samples, respectively: sperm motility (37.2 ± 13.0% and 62.2 ± 15.6%; P = 0.002), normal morphology (60.1 ± 12.2% and 75.5 ± 9.7%; P = 0.006), percentage live sperm (55.8 ± 16.0% and 73.6 ± 12.9%; P = 0.03), HOS (33.7 ± 7.2% and 48 ± 9.7%; P = 0.001) and sperm DNA fragmentation (12.3 ± 4.4% and 5.6 ± 4.4%; P = 0.004). When the Fertile Plus Sperm Sorting Chip was used in Experiment 2 to separate frozen-thawed semen from various sources, improvements were noted between the original and sorted samples, respectively, with increased motility (22.0 ± 13.0% and 57.0 ± 11.6%; P = 0.0009), normal morphology (58.4 ± 9.6% and 74.0 ± 10.3%; P = 0.005), a higher percentage of live sperm (55.5 ± 11.2% and 68.3 ± 14.2%; P = 0.04), and decreased sperm DNA fragmentation (22.3 ± 14.7% and 8.2 ± 8.3%; P = 0.004); no effect was observed on HOS (21.2 ± 6.0% and 24.9 ± 11.5%; P = 0.19). Our results demonstrate that use of the Fertile Plus Sperm Sorting Chip resulted in a subpopulation of sperm with improved quality parameters. Separation of sperm using a microfluidic device has the potential to select sperm with desirable characteristics for equine assisted reproductive techniques.

On their long path through the female reproductive tract to the fertilization site, spermatozoa are exposed to diverse influences and hazards of the cervical, uterine, and oviducal environment that naturally select viable sperms for the following fertilization. Consequently, this results in a reduction from several billions of sperms in the ejaculate to a functional sperm reservoir within the range of 102 in the isthmus of the Fallopian tube. A technique to deposit spermatozoa directly into the ampulla, thus bypassing most of the reproductive tract, enables a rigorous reduction in number of sperms deposited. Furthermore, it provides a direct assessment of sperm fertility. The aim of our study was to establish an endoscopy-assisted intratubal insemination technique using different sperm dosages, fresh or cryopreserved, to determine adequate conditions for optimal fertilization. Eighteen Simmental heifers were inseminated with fresh semen, and 9 heifers were inseminated with frozen semen using this novel technique. The heifers were synchronized using a modified Ovsynch protocol, and insemination was conducted 18 to 20 h after the second gonadotropin-releasing hormone application. Insemination of heifers was performed under epidural anaesthesia. A tubing system bearing the endoscope and an insemination device was introduced through the vaginal wall into the peritoneal cavity. The insemination device consisted of a tube connected to a curved glass capillary tube loaded with semen. After a visual examination of the ovaries for the presence of an ovulatory Graafian follicle, the capillary tube was inserted directly via the infundibulum into the ipsilateral ampulla and the semen dose was deposited. The entire procedure took ~10 min. Two days later the oviduct was flushed by the same technique. A tubing system connected to a metal catheter served for flushing the embryos and unfertilized oocytes from the oviduct into the uterine horn. Afterward, embryos and oocytes were collected by flushing the uterine horn using an embryo flushing catheter and an embryo filter (EmCon). Embryos were stained using a Hoechst dye to visualise the numbers of attached spermatozoa to the zonae pellucidae. From 18 inseminations with fresh semen doses of 7 to 28 million sperms, 7 embryos at the 2- to 8-cell stage were found. Two of these embryos had more than 10 accessory sperms (AS), 3 had 3 to 6 AS, and 2 were without AS. From 9 inseminations with frozen semen doses containing 1.5 million sperms, we obtained 2 embryos, one at the 4-cell stage without AS and one at the 8-cell stage with 5 AS. Additionally, 3 unfertilized oocytes were collected. In conclusion, these preliminary results demonstrate a promising technique for intratubal AI, which has to be further optimized by studying numbers and treatment of spermatozoa and time of insemination.

Generally, laparoscopic intrauterine insemination (LII) provides a higher success rate than that of cervical insemination in goat. However, the sperm distribution following LII in goat remains unknown, especially when frozen semen is used. This study aimed to evaluate the distribution of frozen-thawed goat sperm after LII. In experiment 1, the frozen-thawed sperm were stained with CellTrackerTM Green CMFDA (CT-Green) or CellTrackerTM Red CMPTX (CT-Red), and then evaluated in vitro for viability and motility at 0, 3, 6, and 9 h after staining. In experiment 2, the CT-Green and CT-Red labelled sperm (30 × 106 sperm in 0.125 mL in each side) were laparoscopicaly inseminated into the left and right uterine horns, respectively (n = 4). After ovariohysterectomy at 6 h post-insemination, the distributions of green and red coloured sperm were assessed by tissue section and sperm flushing. The comparison of semen parameters among groups (control, CT-Green, and CT-Red) were statistically analysed by a general linear model. The results revealed that the fluorochromes used in this study did not impair the sperm motility and viability (P > 0.05). The frozen-thawed goat sperm transuterine-migrated following LII, as both CT-Green (left insemination) and CT-Red labelled sperm (right insemination) were found in both sides of the reproductive tracts. This study concludes that LII in goat would be simpler and consume less time if the semen was unilaterally deposited. Supported by a grant from Chulalongkorn University Centenary Academic Development Project and RGJ-PhD-industrial link program, Thailand Research Fund (PHD/0156/2550).

Long-term preservation of mammalian spermatozoa is regarded as being essential for the conservation of endangered species and for assisted reproduction. Although the method generally applied for semen preservation is storage at –196°C, liquid nitrogen and special equipment might not be available in every situation. Additionally, logistical difficulties make cryopreservation difficult under extreme conditions, such as in the wilderness. Fortunately, with the development of intracytoplasmic sperm injection (ICSI), preserved spermatozoa do not need to maintain their motility. The aim of the study was to investigate alternative preservation methods for spermatozoa without freezing in liquid nitrogen and to examine the effects of the preserved sperm on fertilization after injection into in vitro-matured pig oocytes. The pig was chosen as the model for investigations, and the alternative preservation methods included heat-drying, flame-drying, or freezing (–20°C, household refrigerator) of spermatozoa. After semen collection and swim up, sperm concentrations were adjusted to 0.5 to 1 × 105 cells mL–1. For heat-drying, aliquots of sperm suspension (50 µL) were dehumidified at 50, 56, or 90°C each for 45 min, or at 120°C for 20 min. Flame-drying was performed by flaming 5 µL of semen suspension or sperm-rich fraction for up to 2 s on glass slides. Dried semen samples were stored at 4°C. Further, sperm samples were frozen (100 µL/tube) without cryoprotectant and kept at –20°C. The specimens were stored up to 5 days. Before ICSI, heat-dried and flame-dried samples were rehydrated with 50 and 10 µL of ultrapure water (Millipore®, Millipore Corp., Billerica, MA, USA), respectively. Frozen sperm were thawed for 10 min at room temperature. Afterward, spermatozoa were injected into in vitro-matured pig oocytes at metaphase II, and sperm-injected oocytes were activated artificially by 10% ethanol. Subsequently, in vitro culture in mTCM-199® [20 µg mL–1 of insulin, 0.08 mg mL–1 of l-glutamine, 50 µg mL–1 of gentamicin, and 20% FCS (vol/vol)] with 10 µg mL–1 of cycloheximide was performed for 24 h. Afterward, oocytes were fixed, stained (aceto-orcein staining), and investigated for signs of fertilization. Positive fertilization criteria included extrusion of the second polar body, formation of two pronuclei, and the presence of a visible sperm tail. The fertilization ability of heat-dried samples achieved 9.0% (6/67), 8.8% (6/68), 2.7% (2/75), and 0% (0/60) at 50, 56, and 90°C for 45 min and 120°C for 20 min, respectively. Flame-drying reached a fertilization rate of 14.0% (6/43) v. 4.4% (3/69) for the sperm-rich fraction v. swim-up spermatozoa, respectively. With frozen semen (–20°C), 9.4% (13/138) fertilized oocytes were obtained. These results imply that ICSI with spermatozoa, which were alternatively preserved at various temperatures and circumstances in combination with artificial oocyte activation, may fertilize the oocytes. The best results were accomplished with the flame-dried sperm-rich fraction.

Recently, a new procedure for the analysis of sperm DNA fragmentation has been developed for human and different mammalian species (Sperm-Halomax®), based on the sperm chromatin dispersion test (SCDt); however, no studies has been performed specifically on canine frozen–thawed-stressed semen but is there for cooled semen. The aim of this work was to assess the effect of a stressor (24 h in an oven at 38°C) on canine frozen–thawed semen using the SCDt to resemble what happens in the female reproductive tract. For this purpose, ejaculates were collected by digital manipulation from 4 healthy beagle dogs and the sperm-rich fraction of the ejaculates from 3 different dogs was pooled each time. All the pooled semen samples (n = 4) used presented physiological values concerning to routine semen parameters (motility, morphology, and sperm concentration). After evaluation, semen samples were centrifuged and the sperm pellet resuspended to a final concentration of 100 × 106 sperm mL–1 in 2 steps with CaniPRO Freeze (Minitub, Tiefenbach, Germany). Sperm were slowly cooled to 5°C and then loaded into 0.5-mL plastic straws. After that, straws were frozen in liquid-nitrogen vapours for 10 min and stored into a nitrogen tank. Straws were thawed in a water bath (30 s/37°C) and incubated for 24 hours at 38°C before analysis. The sperm DNA fragmentation was assessed in fresh semen and frozen–thawed-stressed samples using the Sperm-Halomax® commercial kit specifically developed for canine semen (Halotech DNA SL, Madrid, Spain) following the manufacturer’s instructions. Slides were stained for green fluorescence staining and 500 sperm per slide were counted using fluorescence microscopy. The sperm DNA fragmentation index (%) was compared between fresh and frozen–thawed-stressed semen samples by ANOVA. Results were expressed as mean ± standard error of the mean. The results obtained showed that subjecting thawed semen to 24 h in an oven at 38°C significantly increased (P

The decrease in the fertility of males can have a negative economic impact on the production of milk and meat in cattle. As a result, interest exists in evaluating diverse proteins that might be able to increase the fertility of sperm. This is the case of the heparin-binding proteins (HBP), specifically the fertility-associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), which act to favour capacitation and the acrosome reaction, perhaps by modulating the immune system response toward the sperm. The objective of this study was to evaluate the effect of adding recombinant FAA and recombinant TIMP-2 to Hereford sexed semen (X-chromosome-bearing sperm) for use in an IVF program. For IVF, X-sorted semen was prepared both with and without HBP. For this, 25 μg of FAA and 25 μg of TIMP-2 were added per milliliter of semen and were left to adsorb for 20 min. The semen was then diluted, packaged in straws and cryopreserved. Ovaries were obtained from a slaughterhouse and transported to the laboratory to obtain cumulus–oocyte complexes by follicular aspiration and were cultured in maturation medium (TCM-199 supplemented with FSH, LH and oestradiol) for 24 h. All incubations were performed at 38.5°C in a humidified atmosphere of 5% CO2 in air. For insemination, frozen-thawed semen was washed by centrifugation in 2 concentration gradients of a silica-based colloid solution. The sperm concentration used for IVF was 1 × 106 spermatozoa mL–1. Gametes were co-incubated for 22 h in fertilization medium (TALP added with BSA, heparin, penicillamine, hypotaurine and epinephrine). For IVD, presumptive zygotes were incubated for 7 days in a modified IVD medium (SOF) supplemented with FCS and BSA. A total of 363 in vitro-matured oocytes were used in the control vs 405 oocytes in the treatment group. A Pearson chi-square test was used to determine the statistical significance. The cleavage rates for the control (61.4%) and treatment (83.7%) groups were different (P 0.1). The fact that there was an increase in the early embryonic development rates and not in the development of the embryos to the blastocyst stage suggests that HBP could play a role in fertilization, but not in embryo development. The results also demonstrate that the addition of the recombinant proteins to the semen increased the fertilizing capacity of the sperm at a concentration of 1 × 106 sperm cells mL–1 of fertilization medium. To further elucidate the effect of the HBP, an evaluation of sperm penetration and pronuclei formation is proposed as a complement to these results. We thank Tod C. McCauley, Roy L. Ax and the staff at TMI Laboratories International Inc. for their assistance. We also thank Richard W. Lenz and the staff at Sexing Technologies for their assistance.

No studies have investigated the IVF rate of South African indigenous Kolbroek sperm cells following cryopreservation. The objective of this study was to test if frozen–thawed Kolbroek pig sperm cells could penetrate pig oocytes matured in vitro. Pig ovaries were collected from a local abattoir and cumulus–oocytes complexes were obtained by aspiration and were then in vitro matured in TCM-199 supplemented with 10% pig follicular fluid, 10% fetal bovine serum, and 1 μg mL–1 of FSH and LH. Following 44 h of incubation, 200 matured pig oocytes were randomly assigned to 2 treatments with frozen–thawed and fresh (control) Kolbroek pig sperm cells. For IVF, Kolbroek sperm cells were in vitro capacitated using Brackett and Oliphant’s sperm wash medium. Matured pig oocytes and sperm cells were co-incubated for 24 h in Brackett and Oliphant’s IVF medium. Following fertilization, presumptive zygotes were in vitro cultured at 39°C in 5% CO2, 5% O2, and 90% N2. Rate of fertilization was identified by the number of cleaved zygotes. Data were analysed by ANOVA. The total motility of Kolboek pig sperm cells used for IVF was 40% for frozen–thawed sperm cells and 80% for fresh sperm cells. The results showed that Kolbroek pig sperm cells were able to penetrate pig oocytes in vitro. However, no significant (P

Sorting of sperm by flow cytometer has allowed selection of offspring of predetermined sex in several species by artificial insemination, although the success rate is often lower than with non-sexed semen. In horses, the problem was partially overcome with hysteroscopic insemination using sex-sorted fresh sperm. However, when sex-sorted frozen–thawed sperm were used the pregnancy rate was heavily reduced in comparison with non-sexed frozen–thawed semen. Because it has been demonstrated that in vitro assisted reproductive techniques, namely intracytoplasmic sperm injection (ICSI), has permitted live foals to be obtained using sperm with low fertility in the field, in this study we investigated the possibility of using ICSI with sexed-sorted frozen–thawed sperm for equine embryo production in vitro. Briefly, semen was collected from two Standardbred stallions of proven fertility (Stallions A, B), sorted using a MoFlo SX flow cytometer and frozen (Johnson LA and Welch GR 1999 Theriogenology 52, 1323–1341). Sex-sorted and control non-sexed frozen semen (two stallions of in vitro proven fertility: C, D) was thawed, centrifuged on a Percoll gradient, washed and diluted 1:1 in PVP before ICSI. Oocytes were collected from ovaries of slaughtered mares and matured in vitro. Metaphase II oocytes were injected with sperm, subsequently cultured up to the blastocyst stage and frozen conventionally in 10% of glycerol (Galli et al. 2002 Theriogenology 58, 713–715). Six embryos from sexed-sorted sperm were thawed and non-surgically transferred in naturally cycling synchronous recipient mares. Results are summarized in Table 1. Overall, 70 and 58 (stallion A, B) and 30 and 15 (stallion C, D) oocytes were injected with sex-sorted or control frozen–thawed sperm, respectively. Mean cleavage rates were 20.3% for sorted sexed sperm and 71.1% for control, showing a significantly lower cleavage rate for sexed sperm. This difference was reflected in the number of blastocysts obtained (4.7% v. 20.0%). From the 6 frozen–thawed embryos derived from sexed sperm, that were transferred, 4 pregnancies resulted. One pregnancy was lost around 21 days, a second was pharmacologically aborted, and two were maintained (one from male and one from female sorted semen are currently in the 11th month of gestation). In conclusion, this study demonstrates that ICSI with sex-sorted sperm can be used for producing equine blastocysts able to establish pregnancies at a high rate following embryo transfer. However, the overall efficiency of the system is limited due to the very low cleavage rate obtained with sexed-sorted frozen–thawed sperm. Table 1.Development of embryos produced by ICSI with sorted and non-sorted frozen–thawed semen This work was supported by an RFO (ex 60%) and Camera di Commercio Cremona grant. The Authors wish to thank Società Italiana Produttori Sementi.

In the present study, we investigated handling, activation and assessment procedures for cane toad (Bufo marinus) spermatozoa. Optimisation of these techniques will facilitate the maintenance of sperm viability during cryopreservation and during in vitro fertilisation (IVF) techniques in reproduction technologies for endangered species. Spermatozoa were taken from testicular macerates and assessed using plasma membrane integrity assays (live/dead stains) and quantitative scores of motility parameters. In the assessment of sperm viability using live/dead stains, there were small but significant differences in the percentage of sperm from cryopreserved samples staining positive with propidium iodide, Hoechst H33258 and Trypan blue; these differences were not large and all stains performed acceptably. Spermatozoa were activated by dilution of testicular macerates in water at one of two dilution ratios (1 : 6 or 1 : 20) with or without 0.1–5.0 mm theophylline. Sperm plasma membrane integrity (unstained spermatozoa) was unaffected by either dilution ratio (osmolarity) or theophylline concentration. However, sperm motility was significantly affected by osmolarity and theophylline concentration. The stimulation of sperm motility increased with higher theophylline concentrations and these strongly interacted with lower osmolarities through a higher dilution ratio of sperm macerates with water. Spermatozoa were exposed to increasing centrifugation forces to determine tolerance to physical stresses encountered during washing procedures. Forces between 50 and 800g were associated with a significant reduction in motility (mean 56 ± 3% decreasing to 27 ± 3%), but did not affect staining. In conclusion, centrifugation should be minimised in anuran sperm washing procedures; osmotic shock associated with higher dilution ratios reduces the capacity of anuran sperm to achieve high percentages of motile sperm, leading to a likely trade-off between dilution required for activation and sperm motility to optimise IVF fertilisation rates; and optimal conditions for sperm motility after activation occur at lower dilutions of suspensions with 5.0 mm theophylline. The present study has improved protocols for the handling of anuran sperm during pre- and post-cryopreservation procedures.

The substantial decrease of sperm quality after cryopreservation remains an important issue in the artificial insemination industry. Sperm coating with Triladyl® (Minitübe, Tiefenbach, Germany) during ejaculation can preserve sperm characteristics and oocyte penetrating capacity of fresh bovine spermatozoa stored in egg yolk diluent for up to 6 days (De Pauw et al. 2003 Theriogenology 59, 1109–1122). Since collecting semen in a tube containing egg yolk-Tris extender (sperm coating) limits the period of contact between spermatozoa and seminal plasma, the present experiment was conducted to assess if this slightly adjusted method of sperm collection could also have a significant effect on bovine sperm quality after cryopreservation. Semen of five young Holstein Friesian bulls was collected by means of an artificial vagina connected to an empty tube (Group 1; five ejaculates per bull) or a tube containing 4 mL of an egg yolk-Tris extender (Groups 2 and 3; each five ejaculates per bull). The semen samples of Group 1 were conventionally diluted in straws (60 × 106 sperm/mL), frozen, and stored in liquid nitrogen. The samples of Group 3 were centrifuged, and after removing diluent and seminal plasma, the sperm pellet was conventionally diluted and processed. The samples of Group 2 were processed without removal of the supernatant. After thawing each ejaculate was analyzed for average path velocity (VAP), beat cross frequency (BCF), and progressive motility (PROG) using CASA (Minitübe, Tiefenbach, Germany). Furthermore, the membrane integrity of each sample was evaluated using fluorescent SYBR®–14/PI staining (BD Biosciences, Erembodegem, Belgium). All parameters were compared among the three groups of sperm using univariate analysis of variance (SPSS 12.0; SPSS, Inc., Chicago, IL, USA). No significant differences could be observed among the three groups for all of the evaluated sperm characteristics (Table 1). A significant effect of the bull could be determined for all analyzed parameters (P ≤ 0.02), except for the percentage of moribund cells. Nevertheless, the group-bull interaction was never statistically significant. Coating bovine sperm with an egg yolk-Tris extender during ejaculation cannot prevent the substantial deterioration of the spermatozoa that occurs during freezing and thawing since this method of sperm collection does not significantly influence the motility parameters or the membrane integrity after thawing. Table 1. VAP, BCF, PROG, and percentage of membrane-intact, dead, and moribund spermatozoa for the three groups of sperm This research was supported by IWT (no. IWT/020727).

Preincubation of semen before insemination may alter the sex ratio of resulting embryos (Lechniak, D. et al., 2003 Reprod. Dom. Anim. 38, 224–227). The overall objective of this study was to evaluate effects of aging sperm before insemination for altering the sex ratio of bovine embryos. In the first experiment oocytes presumed mature were inseminated with frozen semen within minutes post-thaw and percoll-preparation (control) or after aging for 14h post-thaw in a 34.4°C water bath, or after aging for 23h post-thaw at 4°C. Sperm from 4 different bulls, representing 3 different breeds, were utilized (1 bull per experimental replicate). Sperm motility was assessed after aging and percoll preparation. Zona pellucidae were removed from cleaved embryos (43–68h post-insemination; hpi) using 0.5% pronase. Blastomeres were dissociated and counted before transfer to PCR tubes. Sex of cleavage-stage embryos was determined using Ampli-Y™(Bredbacka, P. 1998 Reprod. Nutr. Dev. 38, 605–613). Data were analyzed using a randomized block design with mixed models of SAS (2000) after testing for normality. Aging semen for 14h post-thaw reduced the proportion of motile sperm and compromised the ability of presumptive zygotes to cleave after insemination (Table 1). However, ability of cleaved embryos to develop to the 8–16-cell stage was not affected. Number of blastomeres comprising cleavage-stage embryos that resulted from insemination of oocytes with aged semen was similar to that for controls. Insemination of oocytes with semen aged 14h in a water bath increased the proportion of female embryos (Table 1).To determine if effects of aging semen on altering the sex ratio of bovine embryos was time dependent, oocytes were inseminated with frozen semen within minutes post-thaw (control) or after aging for 8 or 14h post-thaw in a 34.4°C water bath. Sperm from 3 different bulls, representing 2 different breeds were utilized (one bull per experimental replicate). The experiment was replicated 3 times with 195–233 oocytes inseminated within each treatment. Sperm motility averaged 72.7, 65.7 and 45.0% for control and semen aged for 8 and 14h, respectively (SEM=10). Cleavage of inseminated oocytes (67hpi) was similar regardless of sperm treatment (68.5, 70.2 and 70.1%; SEM 14.6, for control semen or after aging for 8 or 14h post-thaw, respectively). Insemination of oocytes with semen aged for 14h tended to increase the proportion of female embryos (51.6 v. 38.3 and 39.1%; sperm aged for 14h v. 8h or control, respectively;; P=0.08; SEM=9.7). Within the seven replicates across both experiments, the differences in percent female for control v. aged were 5.2, 6.3, 14.9, 18.5, 20.0, 29.7 and 60%, with the highest three being significantly different (P

Research from 1930 to 1992 is reviewed with regard to storage of semen at reduced (0-5 degrees C or 10-15 degrees C) and at ambient temperatures. Diluents investigated have included synthetic buffers combined with sugars and egg yolk or its fractions, milk from various sources, glycine, and other substances. Irrespective of the diluent, dilution rate, temperature or conditions of storage, the spermatozoa deteriorate with time of storage. Changes include reduction in motility and morphological integrity of spermatozoa, accompanied by a decline in their survival in the female reproductive tract, reduction in fertility and increased embryonic loss. In critical studies, fertility declined rapidly when semen stored for more than 24 h was used for cervical insemination, but after intrauterine insemination some spermatozoa maintained their fertilizing capacity up to 10 days. Laparoscopic intrauterine or transcervical inseminations could be the means of improvement of fertility. These methods may eliminate the problem of sperm transport through the cervix and ageing of spermatozoa in the reproductive tract, thereby improving fertilization of ova and reducing embryonic loss.

Artificial insemination (AI) allows individual bucks to be exploited widely and so is a potentially useful tool for the rapid genetic improvement of fibre goats. In Australia, where there is a desire by farmers to improve the productivity of their goats, AI may best be adopted under extensive grazing conditions using control of ovulation to allow efficient and accurate timing of the deposition of frozen-stored semen. Although ovulatory activity is influenced by the manipulation of environmental factors, the time of ovulation is synchronised most accurately by the combined use of intravaginal progestagens and pregnant mares' serum gonadotrophin. However, the costs of these exogenous hormones remain high, which justifies investigation of alternative methods to control ovulation. Bucks show strong seasonality in the quality and quantity of their sperm production, and so there is limited time in which semen may be collected for storage and AI, but this can be extended by optimising nutrition and management. There appears to be no improvement in the fertility of stored semen when seminal plasma, which contains egg yolk coagulating enzyme, is removed and an extender containing only a low concentration of egg yolk is used for dilution. Simple methods have been developed for 1-step dilution and freezing of buck semen. However, the post-thawing viability of spermatozoa frozen in pellets on dry ice is higher than for semen frozen in straws in liquid N2 vapour, although straws are preferred for commercial trade. For frozen-thawed semen, fertility after laparoscopic insemination is high, whereas the fertility after cervical insemination is considerably lower but improves by the deeper placement of semen into the reproductive tract. Does are best inseminated 5-10 h before the expected time of ovulation. A dose as low as 1 x 106 motile spermatozoa may be used for laparoscopic insemination of thawed semen that was previously diluted at rates (semen: diluent) of 1:2 to 1:23. However, for the cervical method, a low dilution rate of 1:2 allows a sufficiently small, highly concentrated dose of at least 120 x 106 motile frozen-thawed spermatozoa t o be deposited into the reproductive tract of the doe. Cervical insemination is cheaper and simpler than the laparoscopic method, and this warrants the development of an improved technique for the consistent, deep deposition of frozen-thawed semen through the cervix in a high proportion of does.

The experiments, using a total of 1833 Cashmere does, were conducted to examine the effects on fertility and fecundity of intravaginal treatment for control of ovulation, time of insemination by cervical and laparoscopic methods, semen processing in pellets and straws, and number of frozen-thawed motile spermatozoa. Adult does (greater than or equal to 2.5 years) that had previously kidded were inseminated into the cervix, and maiden (kid: 6-8 months; hogget: 18 months) and adult does were inseminated laparoscopically. The rates of pregnancy determined by ultrasonic scanning (75-80 days after insemination) for cervical (Experiment 1) and laparoscopic (Experiments 2 and 3) insemination were 39.1%, 63.6% and 52.1%. The number of motile spermatozoa did not affect fertility after cervical (80, 120 and 160 x 10(6) or laparoscopic (Experiment 2: 15, 30 and 60 x 10(6); Experiment 3:5, 10 and 20 x 10(6) insemination. The method of insemination or number of motile spermatozoa did not affect fecundity. With cervical insemination, fertility and fecundity improved with increasing depth of insemination into the reproductive tract. Intravaginal sponges containing fluorogestone acetate and controlled internal drug release (CIDR) devices containing progesterone were equally effective for control of ovulation when combined with an injection of pregnant mare's serum gonadotrophin (PMSG; 200 i.u.) before insemination. Fertility was higher when does were inseminated before than after ovulation. After laparoscopic insemination, results were similar for semen frozen as pellets and in straws, and for 3-, 6- and 24-fold pre-freezing dilution of semen. For kid, hogget and adult does in Experiment 3, pregnancy rates were 34.0%, 55.4% and 63.9% and rates of fecundity were 1.17, 1.22 and 1.27 fetuses/pregnant doe.

The fertility of Merino ewes artificially inseminated with semen diluted tenfold in milk or buffered glucose solution was lower than that of a control group of ewes inseminated with the same number of spermatozoa in undiluted semen. By means of centrifugation, the concentration of spermatozoa in the insemination dose of diluted semen was raised to match that of the undiluted semen and then the effect of dilution on fertility was eliminated for the glucose-diluted semen, but not for the milk-diluted semen. Respective percentages of ewes not returning to oestrus and ewes lambing were, after insemination with undiluted semen, 60.5, 46.7; with milk-diluted semen, 55.9, 40.2 and after reconcentration 56.0, 38.0; with glucose-diluted semen, 48.2, 35.3, and after reconcentration 62.1, 46.1. In another experiment, the percentage of ewes lambing after insemination with undiluted semen, with semen diluted tenfold with glucose, and with semen diluted with a ribose mixture and chilled at 5°C for 24 hr were respectively 67.0, 58.0, and 26.1. Both diluents contained 6% (v/v) egg yolk and diluted semen samples were reconcentrated to the original sperm density of the semen immediately before insemination.

Ewes were artifically inseminated with semen diluted in skim milk. In the first experiment, with an insemination dose of 50 x 106 spermatozoa, 10-fold dilution of semen did not reduce the fertility of the diluted sample if it was reconcentrated by centrifugation to the same volume as the undiluted controls. However, three later experiments conducted under extensive pastoral conditions showed that dilution in milk lowered the fertility of semen, and reconcentration of the spermatozoa had no effect. Only 8.8% fertility followed the insemination of 50 x 106 spermatozoa in semen diluted 10-fold and stored at 5°C for approximately 36 hr. The administration of 50 or 200 mU of synthetic oxytocin to ewes at an early stage after insemination had no effect on fertility. The intensity of marking with the crayons carried by vasectomized teaser rams, thought to be a measure of the intensity of display of oestrus, was positively correlated with the fertility of ewes at insemination. The greater the consistency and opacity of the vaginal mucus at the time of insemination, the lower was the fertility of the ewe. The fertility of ewes was highest during approximately the first 12 hr of oestrus, and dropped progressively through the two subsequent 12 hr periods.