An effective method for freezing White Italian gander semen

Efficiency of freezing method, worked out for the White Italian gander semen was evaluated by comparing motility, morphology and fertilizing ability of spermatozoa in fresh and frozen-thawed semen. A part of pooled semen, collected from 25 White Italian ganders by dorso-abdominal massage was used immediately for artificial insemination of 10 geese (the control group) with a dose of 80 microl. This insemination was performed six times at weekly intervals. The remainder of the semen was diluted 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with 6% (v/v) of dimethylformamide (DMF) and frozen to -140 degrees C at a rate of 60 degrees C/min. Frozen semen was thawed in a 60 degrees C water-bath and inseminated twice weekly in a dose of 100 microl (10 females of the experimental group, 12 inseminations were made). The freezing process affected spermatozoa motility and morphology, but had no effect on their fertilizing ability. Positive movement was observed in 50-60% of the spermatozoa in fresh semen and about 40% of the frozen-thawed cells. The average percentage of total live and live normal spermatozoa decreased due to freezing from 92.2 to 68.4% and from 34.7 to 14.1%, respectively. After the fresh semen insemination with average 12 million of the live normal spermatozoa per week average fertility was 88.24%; hatchability of set eggs was 80.88% and hatchability of fertile eggs was 91.67%. For frozen-thawed semen inseminated with average 9.5 million of the undamaged spermatozoa per week, the average fertility and hatchability rate was 83.78, 73.87, and 88.17%, respectively. Fecundity rates obtained after insemination with the frozen-thawed gander semen allow for the application of the freezing technique into breeding practice, in place of natural mating or to assist natural mating in periods of lowered fertility level.     

Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males

This study aimed to establish artificial insemination (AI) protocols to predictably initiate pregnancy during the breeding season in the European brown hare (EBH) (Lepus europaeus PALLAS, 1778). Semen was collected from seven captive and eight free-ranging males by means of electroejaculation. Semen from the free-ranging males was cryopreserved using directional freezing. Total motility/integrity of fresh and frozen-thawed semen was 91.6%/87.7% and 46.9%/53.8%, respectively. Ovulation was induced in ultrasonographically preselected females using a gonadotropin-releasing hormone analogue. Each female was inseminated with 1 mL fresh (Group A, n = 16) or frozen-thawed semen (Group B, n = 9) at a concentration of 100 × 10⁶ spermatozoa/mL. The use of ultrasonography (10 to 22 MHz) confirmed the intracervical semen deposit, the success of artificial ovulation induction (formation of postovulatory corpus luteum), and permitted the monitoring of individual pregnancies. Although sperm motility/integrity was significantly different between groups, no significant difference was detected in conception rates (A, 87.50%; B, 77.78%). Because of embryonic resorption, there was a slight difference in fertility rate between groups (A, 62.5%; B, 77.78%). Overall, AI in captive EBH using fresh and frozen-thawed semen achieved successful fertility rates. Long-term cryopreserved semen was used to bring new genetic material from the wild into a genetically limited captive population without extensive animal transport. Therefore, AI has the potential to enhance breeding programs for EBH especially when cryopreserved semen from wild donors is used.     

The effect of sperm number on fertility in blue fox vixens (Alopex lagopus ) artificially inseminated with frozen silver fox (Vulpes vulpes ) semen

During the breeding seasons of 1989 and 1990, a total of 617 blue fox vixens aged 1 to 6 years (mean +/- SEM, 2.6 +/- 0.1) were inseminated with frozen silver fox semen with either 150 million (n = 213, 1989 + 1990), 100 million (n=172, 1990), 75 million (n = 119, 1989) or 37.5 million (n = 113, 1989) spermatozoa per insemination. Two intrauterine inseminations, each with an insemination volume of 1.0 ml, were performed at 24-hour intervals on the first and second days after maximum vaginal electrical resistance was measured. Conception rates were 87% (186 of 213) with 150 million spermatozoa per insemination, 85% (146 of 172) with 100 million, 77% (91 of 119) with 75 million and 68% (77 of 113) with 37.5 million. The mean numbers of cubs per litter +/- SEM for the four groups were 7.6 +/- 0.2 (168 registered litters), 7.5 +/- 0.3 (115 litters), 6.4 +/- 0.4 (86 litters) and 6.4 +/- 0.4 (75 litters). A negative effect on both the conception rate and mean litter size at whelping was observed with decreasing sperm numbers (conception rate percentage: p = 0.0001, Chi-square, litter size: p = 0.02, Kruskal-Wallis Test). Only the two larger numbers of spermatozoa gave litter sizes comparable to those obtained by artificial insemination (AI) with fresh semen.     

The possibility of obtaining intergeneric hybrids via White Kołuda (Anser anser L.) goose insemination with fresh and frozen-thawed Canada goose (Branta canadensis L.) gander semen

The objective of the present experiments was to produce the intergeneric hybrids of domesticated and wild goose via artificial insemination with fresh and frozen-thawed semen. The experiments were carried out during two successive goose reproductive seasons, on eight five-year-old Canada Goose (Branta canadensis L.) males used as semen donors and 16 two-year-old White Ko?uda geese designated to fertility tests. Pooled semen was collected twice a week by the dorso-abdominal massage. In freshly collected semen, ejaculate volume, color, consistency, degree of fecal or blood contamination, spermatozoa concentration, motility, and morphology were evaluated. Part of the semen collected in the first year of the experiment (Experiment 1) was used for geese insemination with fresh semen, while the remainder was frozen. In Experiment 2 all samples were subjected exclusively to freezing procedure. Geese were inseminated once a week with fresh semen in a dose of 80 μl or 160 μl, and twice a week with frozen-thawed semen in a dose of 80 μl (160 μl per wk) or 100 μl (200 μl per wk). Eggs were set weekly and incubated up to hatching.The volume of ejaculates varied from 0.100 to 0.470 ml; spermatozoa concentration from 140 to 310 million ml⁻¹; progressive movement was observed in 40 to 60% of spermatozoa; the percentage of total live spermatozoa ranged from 69.3 to 92.0%, the highest percentage (34.0-68.3) was represented by live normal spermatozoa and those with bulb-head (13.3-41.0). Cryopreservation caused a decrease in percentage of motile cells to 30%; total live spermatozoa contribution by 27.2%p, including those live normal by 15.9%p (in relation to the fresh semen), bulb-head spermatozoa by 10.9%p, and increase (by 5.9%p) in number of spermatozoa with other deformations. Goose insemination 1×/week with fresh semen containing about 10.3 million live normal spermatozoa resulted in 66.7% of fertile eggs and with dose higher by 2.8 million spermatozoa (on average) the fertility increased by 20.9%p (up to 87.6% on average). Hatchability from set and fertile eggs was 55.9% and 83.9% vs. 66.3% and 75.6%, respectively. After twice a week insemination with frozen-thawed semen containing about 10.2 million live normal cells 58.2% eggs were fertile; hatchability from set eggs was 42.8% and from fertile eggs 71.7%, while insemination dose increase by 2.7 million spermatozoa per week caused a fertilization increase by 3.8%p (62.0% on average), this increase was not statistically significant, but hatchability from the fertile eggs (95.4%), was significantly (P < 0.05) higher.The use of AI with fresh semen in the creation of intergeneric hybrids of Canada goose males and White Ko?uda females allows a high level of egg fertility to be obtained. Furthermore, one limitation which is the short reproductive season of the Canada goose may be overcome by the use of cryopreserved semen.     

Sperm-induced leukocytosis in the equine uterus

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

Comparison of conventional and computer-assisted semen analysis in cockatiels (Nymphicus hollandicus) and evaluation of different insemination dosages for artificial insemination

Many psittacine species are threatened in the wild and also rare in captivity. Therefore, successful conservation and breeding programs are important to save these species. Unfortunately, clutches in conservation programs are frequently infertile. Semen evaluation is beneficial to investigate the causes of infertility and is advisable before artificial insemination (AI). In this study, we analyzed the semen of cockatiels (Nymphicus hollandicus) using two different methods and investigated different insemination dosages for AI. Cockatiels (n = 30) were divided into two groups (group A: nine males; group B: six males). The males in group B were endoscopically sterilized, whereas the males in group A were used as semen donors. In the first part of the study, the semen of males in group A was evaluated by semen analysis. Semen samples were collected by the massage technique and examined using a conventional light microscope and a computer-assisted semen analyzer for comparison. Results demonstrated that the evaluations of motility, progressive motility, and sperm concentration, but not of live/dead ratio, correlated strongly for both methods. However, the results for sperm concentration, progressive motility, and live/dead ratio differed significantly. In the second part of our study, the volume and quantity of spermatozoa of the semen samples were adjusted and used for AI of females of group B. Intravaginal insemination with 250,000 spermatozoa resulted in five of 17 (29%) eggs fertilized; however, intracloacal insemination resulted in only four of 57 (7%) eggs fertilized at 232,000 and 250,000 spermatozoa but none at higher or lower dosages.     

A method of artificial insemination in captive White-tailed deer (Odocoileus virginianus )

Production of fawns by artificial insemination in captive White-tailed deer (Odocoileus virginianus ) has been accomplished by using frozen-thawed spermatozoa. The purpose of this study was to determine if frozen-thawed semen deposited at the posterior face of the os cervix could produce conception. Five hand-raised female White-tailed deer and one hand-raised male White-tailed deer were used over two breeding seasons 1984-1985 and 1985-1986. The vasectomized buck was ued to detect estrus in the does. The does were inseminated with frozen-thawed semen containing at least 100 million live normal cells with a 60% or higher motility. The artificial insemination catheters used in this study worked well, but due to the small size of the cervix, the catheter could only be passed up to the first cervical ring, the site at which the semen was deposited. Over two breeding seasons, nine does were inseminated with frozen-thawed spermatozoa; each doe was inseminated once each estrous cycle at one of the following times: 0, 6, 12, 18, 24 or 30 h. after detection of estrus. Of the nine does inseminated with frozen-thawed spermatozoa, six conceived and carried to term 11 healthy normal fawns, yielding an overall conception rate of 67%.     

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

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

Cryogenic preservation of semen from the Aleutian Canada goose (Branta canadensis leucopareia)

Aleutian Canada geese (Branta canadensis leucopareia) were inseminated with frozen-thawed semen containing 6% or 7% dimethylsulfoxide (DMSO) resulting in 32 fertile eggs and 17 goslings; with 7% DMSO, 19 of 31 eggs were fertile. Beltsville Poultry Semen Extender (BPSE), adjusted to 270 ± 30 mOs and 7.5 ± 0.4 pH, was used to dilute semen samples and the DMSO before cryopreservation. About half of the live spermatozoa in the fresh semen (92.9 ± 2.5% live cells, laboratory studies; 87.3 ± 7.3%, insemination trials) survived the freeze-thaw process (46.7 ± 7.8%, laboratory; 33.3 ± 17.8%, insemination trials). Samples of frozen-thawed semen contained a greater percentage of bent spermatozoa (27.1 ± 8.4% of live cells) than fresh semen (14.4 ± 3.0% of live cells). Fecal- and urate-contaminated semen (a common problem when collecting goose semen) reduced the sperm motility score from 3.2 ± 0.6 to 2.7 ± 0.7 and number of live spermatozoa in frozen-thawed semen from 49 ± 9% to 24 ± 18%. Other variables examined that had less of an effect on semen quality included semen extenders, semen holding temperature, dilution and equilibration, relationship between hour of semen collection and level of semen contamination, and the relationship between season and sperm concentration.     

Elimination of apoptotic spermatozoa from rabbit insemination dose using annexin V associated with the MACS technique. A preliminary study

The aim of this study was to verify whether the separation and elimination of the apoptotic fraction in rabbit semen using a MACS technique may improve sperm fertility potential and consequently rabbit kindling rate. Semen samples from 25 New Zealand White (NZW) rabbit males were collected using an artificial vagina and evaluated using the CASA system for concentration and motility. For artificial insemination the best 11 bucks were chosen based on motility parameters. Their ejaculates were mixed to make a heterospermic pool and routinely diluted in a commercial insemination diluent (MiniTüb, Tiefenbach, Germany) at a ratio of 1:6. Diluted heterospermic spermatozoa were filtered through a Sartorius filter to wash out seminal plasma, re-diluted in binding buffer (Annexin V Microbead Kit, Miltenyi Biotec, Germany) at a ratio of 1:3.66 and divided into two groups: an experimental group intended for MACS separation and control group without MACS separation. Then hormonally treated females of NZW rabbits were inseminated with fresh doses of filtered heterospermic semen (n = 27; 0.5 ml I.D. per female) and MACS separated semen (n=28; 0.5 ml I.D. per female). Separation and subsequent elimination of apoptotic spermatozoa (positive selection) from the insemination dose (after negative MACS selection) was verified under in vivo conditions on the basis of increased kindling rate in the experimental group in comparison with kindling rate in the control group (81.3% vs. 73.8%). In conclusion, elimination of apoptotic spermatozoa by the use of the MACS technique results in a slight improvement in kindling rate of rabbit does.     

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.     

The effect of DMA level on morphology and fertilising ability of Japanese quail (Coturnix japonica) spermatozoa

The effect of different levels (2, 4 or 6%) of DMA (dimethylacetamide) on the morphology and fertilising ability of unfrozen quail spermatozoa was evaluated. Semen was collected from 72 males kept individually in cages and randomly divided into four groups: Group I--control -- fresh undiluted semen (12 males) and three experimental groups (20 males each) - semen diluted 1:1 with Lake's extender and supplemented with 2% (Group II), 4% (Group III) or 6% (Group IV) of DMA (final concentration). Sperm morphology was evaluated at each step of semen preparation, i.e. in fresh and diluted semen, semen supplemented with DMA and semen that remained after insemination. For fertility tests, 36 females were divided into four groups (nine females each). Females in the control group were inseminated with 10 microl of fresh semen, in the experimental groups with 40 microl of diluted semen. Each stage of quail semen treatment had a deleterious effect on sperm morphology. The highest percentage of morphologically normal cells in semen evaluated after insemination, was observed in samples with 2% DMA, and the lowest--in samples with 6% DMA. Semen dilution and DMA addition significantly affected the fertilising potency of spermatozoa. Fertility of eggs collected from the control group (71.5% on average) was significantly higher (P相似文献   

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.     

Effects of semen filtration and dilution rate on morphology and fertility of frozen gander spermatozoa.

Feces, urates or dirt originating from feathers often contaminate gander semen during collection, threatening its fertilizing ability. Seminal plasma used as a diluent has a similar effect, particularly on spermatozoa subjected to cryopreservation or short-term storage under refrigeration. The aim of the experiments was to evaluate the effects on spermatozoa motility, morphology and fertilizing ability after minimizing the influence of the contaminants by semen filtration or dilution prior to freezing. Pooled semen, collected twice a week from 9 White Italian ganders by dorso-abdominal massage, was divided into two parts. One sample was filtered and both were diluted in 1:1 or 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with dimethyl-acetamide (DMA) in the final concentration 6% (v/v) and frozen to -140 degrees C in a computerized freezer, at a rate of 60 degrees C/min. In fresh and processed (filtered, freeze-thawed) semen were examined the spermatozoa motility and morphology, and fertilizing ability for freeze-thawed semen, both for unfiltered and filtered. In freeze-thawed semen no tangible differences due to experimental factors were observed in motility and percent of live spermatozoa in total. On average 35 to 42% of the spermatozoa survived the freezing process, but only 10 to 15% were normal, without any damage visible under the light microscope. The fertility of unfiltered freeze-thawed semen inseminated twice a week in a 0.2 mL dose (about 3 to 5 x 10(6) of live normal spermatozoa each) averaged 66.1% and hatchability of the set eggs 57.1 and 86.5% of the fertile eggs. The fertility obtained after the insemination with semen filtered prior to freezing was lower (64.3%), but hatchability was slightly higher (58.6 and 91.1% of set and fertile eggs, respectively). The duration of fertility for filtered semen was longer than that for unfiltered, 10 days after the last insemination the eggs were still fertile. The fertility results of freeze-thawed gander semen were very promising taking into consideration the small amount of inseminated live normal spermatozoa and it is possible to improve this result by increasing the number of spermatozoa in the insemination dose.     

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.     

Pattern of loss of spermatozoa from the vagina of the ewe

In a series of experiments spermatozoa were inseminated blindly into the vagina of ewes and then recovered at varying times after insemination. Most of the spermatozoa inseminated were lost by drainage through the vulva. The rate of loss was not affected by the motility of spermatozoa or oestrous state of the ewe. Initially after insemination the loss was not rapid with 82% of the insemination 18% of spermatozoa remained and by 12 h 10% remained. Spermatozoa were removed from the vagina during withdrawal of the penis after intromission and the extent of this loss varied between rams and with the volume of semen already in the vagina. Up to half the inseminate was lost in this way when there was 0.5 ml of semen in the vagina but only 11% was lost when the volume of inseminate was 0.1 ml. The unavoidable loss of spermatozoa may influence the quantity available for fertilizing ova.     

Caprine blastocyst development after in vitro fertilization with spermatozoa frozen in different extenders

The feasibility of using frozen-thawed semen in caprine IVF outside the breeding season was investigated. Electroejaculated spermatozoa from a Nubian buck were washed twice and then frozen in skim milk- or in egg yolk-based extenders. Goat oocytes were matured and inseminated by frozen-thawed spermatozoa selected by swim-up. In vitro fertilization was performed in a modified defined medium (mDM), altered experimentally, for 24 h. Embryos were cultured in 50 microL of c-SOF + NEA for 9 d. The percentages of oocytes exposed to heparin-capacitated spermatozoa, (previously cryopreserved in skim milk-based extender) that cleaved, reached morula, blastocyst and expanded blastocyst stages were 82.8, 57.1, 35.7 and 30.0%, respectively. Without heparin treatment the rates for cleavage, morula, blastocyst and expanded blastocyst stages were 44.3, 31.4, 18.6 and 8.6%, respectively. Therefore, heparin treatment was included in sperm capacitation. Use of spermatozoa with BSA in the IVF medium yielded no cleavage. Although extenders containing 8 to 20% egg yolk enabled good sperm motility after cryopreservation, in vitro fertilizing ability was compromised under our conditions. By contrast, semen commercially processed in season in an egg yolk-based diluent remained effective for IVF. The highest proportion of blastocysts resulted from the use of spermatozoa diluted in a skim milk extender, heparin capacitation, and insemination in medium containing lamb serum.     

Oocyte transfer in mares with intrauterine or intraoviductal insemination using fresh, cooled, and frozen stallion semen

The objectives were to compare embryo development rates after oocyte transfer with: (1) intrauterine or intraoviductal inseminations of fresh semen versus intraoviductal insemination of frozen semen; (2) intraoviductal versus intrauterine inseminations of cooled semen. In Experiment I, oocytes were transferred into the oviduct, and recipients were inseminated into the uterus with 1 x 10(9) fresh spermatozoa, or into the oviduct with 2 x 10(5) fresh or frozen-thawed spermatozoa. In Experiment II, semen was cooled to 5 degrees C before intrauterine insemination with 2 x 10(9) spermatozoa or intraoviductal inseminations of 2 x 10(5) spermatozoa (deposited with the oocytes). In Experiment I, embryo development rates were similar (P>0.05) for intrauterine versus intraoviductal inseminations when fresh semen was used (8/14, 57% and 9/11, 82%, respectively). However, embryo development rates were lower (P<0.05) when frozen spermatozoa were placed within the oviduct (1/12, 8%). In Experiment II, embryo development rates were higher (P<0.05) when cooled semen was used for intrauterine (19/23, 83%) versus intraoviductal (4/16, 25%) inseminations. We concluded that intraoviductal insemination can be successfully performed using fresh spermatozoa. However, the use of cooled and frozen spermatozoa for intraoviductal inseminations was less successful, and needs further investigation.     

Birth of the first mithun ( Bos frontalis) calf through artificial insemination

The study describes the standardization of a suitable semen cryopreservation protocol for the first time in mithun (Bos frontalis) and birth of the first mithun calf through artificial insemination. The semen samples were collected from adult bulls through the rectal massage method and cryopreserved in liquid nitrogen using tris-egg yolk-glycerol diluent. The diluted semen samples were packaged in 0.50 ml straws and kept at 5°C for 4 h for equilibration. Following the equilibration, the straws were frozen into liquid nitrogen vapour for 10 min and then plunged into liquid nitrogen for storage. It was observed that the progressive motility (%) decreased significantly (P < 0.01) in cryopreserved semen (43.3 ± 4.1) compared with fresh samples (76.6 ± 3.3). The percentages of live spermatozoa (P < 0.01) and spermatozoa with intact acrosome (P < 0.05) also decreased significantly in cryopreserved semen (54.0 ± 3.3 and 64.6 ± 5.3) compared with fresh samples (79.3 ± 2.6 and 85.3 ± 1.8). Simultaneously, the total morphological abnormality (%) was found to be significantly (P < 0.01) higher in cryopreserved samples (15.46 ± 2.68) than in fresh semen (3.85 ± 0.63). A total of three mithun cows were inseminated using the cryopreserved semen. All the cows conceived following insemination and gave birth to healthy calves. The study revealed that mithun semen can be cryopreserved efficiently using tris-egg yolk-glycerol diluent, which can be further used for artificial insemination.     

Assessment of fresh and stored duck spermatozoa quality via in vitro sperm-egg interaction assay

An in vitro sperm-egg interaction assay was used to measue the quality of duck spermatozoa in fresh and stored semen. The inner perivitelline layer (IPVL), which had been separated from laid duck eggs, was incubated with spermatozoa in vitro. The number of points of sperm hydrolysis in the IPVL in vitro was logarithmically correlated with the fertility of the eggs laid by inseminated females, for both fresh semen (r = 0.85, P < 0.001) and stored semen at 5 degrees C for 24 h (r = 0.84, P < 0.001). After semen storage, the ability of spermatozoa to hydrolyze the IPVL decreased by 67.4% compared with the values for fresh semen, whereas egg fertility and sperm motility decreased by 47.8% and 15.2%, respectively. These results suggest that the in vitro sperm-egg interaction assay accurately reflects the fertilizing ability of fresh and stored duck spermatozoa and detects spermatozoal damage due to semen storage more sensitively than motility or fertility tests.