Some effects of the composition of inseminated semen and the site of its deposition on fertility in Gallus domesticus

It is most probable that during natural copulation the semen of the fowl is ejaculated into a shallow position in the vagina of the hen, but during the commercial application of artificial insemination it is generally considered necessary to evert the distal vagina and deposit semen to a depth of at least 5 cm to produce optimal fertilisation of the succession of eggs laid daily by a female for a week post-insemination. Aspects of the artificial insemination technique in relation to the types of semen that are obtained from the male fowl artificially are re-appraised in relation to their effect on fertility. It was confirmed that a smaller number of spermatozoa (50 × 10⁶) than is normally used in commercial practice (>80 × 10⁶) produced good fertility, even when inseminated within 0.5 cm of the vaginal opening in the cloaca. The results were achieved whether or not glucose was present in the inseminate. When semen was deposited in the cloaca, a better fertilisation rate was obtained if ductus-deferens semen was diluted with transparent fluid, which is produced by tumescent tissue in the cloaca during semen collection. However, the same advantageous effect was shown by dilution with synthetic aqueous fluids with and without glucose. The likely role of transparent fluid during natural copulation is discussed. On the basis of the number of spermatozoa found to maintain good fertility by artificial insemination, only 10 μl semen would be required to be ejaculated into each hen during copulation. This may account for the well-known ability of the male fowl to copulate frequently in a day, because the small volume of semen would be replenished, naturally, very quickly in the ductus deferens.     

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%.     

Seminal collection, seminal characteristics and pattern of ejaculation in llamas

Semen was collected from 10/10 llamas during 26/30 (87%) collection attempts using an artificial vagina mounted inside a surrogate female. For the 26 semen collections, the duration of copulation (mount to dismount) with the artificial vagina was 31.7 +/- 12.0 min (mean +/- SD). Seminal pH was 8.1 +/- 1.1, and seminal volume per collection was 3.0 +/- 1.9 ml. Sperm concentration per collection was 1.0 +/- 0.8 x 10(6) sperm/ml, total number of spermatozoa was 2.9 +/- 3.1 x 10(6), total sperm motility was 23.7 +/- 20.0%, and the percentage of morphologically normal spermatozoa was 39.7 +/- 18.5%. Morphologically abnormal spermatozoa were categorized according to abnormal heads (20.1 +/- 19.9%), tail-less heads (8.7 +/- 8.9%), abnormal acrosomes (12.9 +/- 12.4%), abnormal midpieces (1.0 +/- 3.7%), cytoplasmic droplets (11.1 +/- 12.4%), and abnormal tails (6.6 +/- 12.0%). There were 0.3 +/- 0.3 million motile, morphologically normal spermatozoa per collection: less than 1000 during the first 5 min of copulation, 0.01 +/- 0.01 x 10(6) between 5 and 10 min of copulation, 0.04 +/- 0.08 x 10(6) between 10 and 15 min of copulation, 0.09 +/- 0.21 x 10(6) between 15 and 20 min of copulation, and 0.15 +/- 0.28 x 10(6) between 20 min and the end of copulation.     

Current topics in artificial insemination of sheep

There have been developments in several aspects of artificial insemination (AI) in recent years, some of which have been directly responsible for proliferation of AI in the sheep-breeding industries of several countries. The most notable advances have probably been associated with the development of intrauterine insemination by laparoscopy. There is potential for refinement of some of the related techniques, particularly in the area of control of ovulation and definition of appropriate times and optimum doses of spermatozoa for insemination. It is unlikely that laparoscopic AI will be developed sufficiently that it will become readily affordable, and therefore widely practised, by commercial producers. Unfortunately, there has been little progress in the past few years in improvement of the methods of cryopreservation of ram semen. There is considerable potential for AI to have a significant impact on the genetic improvement of sheep, though this has yet to be evaluated in practice. However, if the full potential of AI in sheep is to be realized, it will likely only happen when methods of freezing semen are improved sufficiently that cervical or even vaginal insemination can be widely used with frozen-thawed semen, or when practicable methods of deep cervical or intrauterine insemination through the cervix are developed.     

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.     

Sources of variation in the reproductive performance of ewes inseminated with frozen-thawed ram semen by laparoscopy

Fertility data from 8 artificial insemination programs, involving more than 5000 ewes and 110 rams in 3 flocks, were analyzed to determine variation due to individual AI program and ram in the reproductive performance of ewes inseminated with frozen-thawed semen by laparoscopy. The semen had been previously frozen by commercial AI centers in either pellets or straws. Both AI program and individual ram affected the proportion of ewes pregnant and the number of fetuses per ewe inseminated, but not the number of fetuses per pregnant ewe. Semen samples from 97 of the rams used were analyzed on a Hamilton Thorn HTM 2000 image analyzer for sperm concentration, percentage of motile and progressively motile spermatozoa, mean progressive velocity, and mean linear index. The correlations between these traits and reproductive performance obtained after insemination were calculated. There was large variation in the quantity and quality of the frozen semen, but only the number of total and motile spermatozoa inseminated per ewe was correlated with fertility (0.25 and 0.26, respectively). Regression analysis showed that none of the traits measured were useful for predicting fertility.     

Sources of spermatozoa loss during collection and artificial insemination of horses

During artificial insemination of horses, it is important to accurately estimate the number of spermatozoa in each insemination dose. However, little research exists regarding sources of spermatozoa loss during collection and artificial insemination. Therefore, spermatozoal losses were quantified in the dismount loss (187.6×10(6)±62.5×10(6)spermatozoa), gel fraction (179.8×10(6)±61.7×10(6)spermatozoa), and the collection receptacle (136.1×10(6)±26.9×10(6)spermatozoa). Spermatozoal losses were examined in the centrifuge tube (25.8×10(6)±2.1×10(6)spermatozoa), AI pipette during the air removal (90.9×10(6)±8.5×10(6)spermatozoa), and spermatozoa remaining in the AI pipette after insemination (342.9×10(6)±21.4×10(6)spermatozoa). The average cumulative loss was 14.2±2.9% of the total spermatozoa ejaculated with approximately half of the loss due to the process of semen collection and half due to the process of artificial insemination. Spermatozoa retained in the AI pipette, after insemination with extended semen, represented the greatest source of loss.     

Conception rates following intrauterine insemination of European (Dama dama dama) fallow deer does with fresh or frozen-thawed Mesopotamian (Dama dama mesopotamica) fallow deer spermatoza

A total of 121 European fallow deer does, being either parous ( n = 15) or nulliparous ( n = 106), were treated with intravaginal progesterone impregnated controlled internal drug release (CIDR) devices for 14 days. The does were divided into three treatment groups and inseminated in utero by laparoscopy, at approximately 65 hours after CIDR device removal, with 25 × 10⁶ fresh Mesopotamian ( n = 40), 25–35 × 10⁶ frozen-thawed Mesopotamian ( n = 41) or 30–32.5 × 10⁶ frozen-thawed European ( n = 40) fallow deer spermatozoa. The semen used had been collected, from two Mesopotamian and two European fallow deer bucks, by electroejaculation under general anaesthesia. Pregnancy was diagnosed by rectal ultrasonogrdphy on Day 50 after insemination.
There were no apparent differences in the quality of ejaculates between the two subspecies of fallow deer. The volume of semen and the total number of spermatozoa ranged between 0.6–1.2 ml and 2.11–4.95 × 10⁹ per ml of semen, respectively. Evaluation of frozen-thawed spermatozoa revealed post-thaw motility rates between 50–70%. The overall conception rate was 65.3%. A higher conception rate was observed following insemination with European than Mesopotamian frozen-thawed spermatozoa (75% vs. 53.7%, respectively, P < 0.05). Insemination with fresh Mesopotamian spermatozoa increased the conception rate to a level not significantly different from that observed following insemination with European frozen-thawed spermatozoa (67.5% vs. 75%, for fresh Mesopotamian and frozen-thawed European semen, respectively).     

Cryopreservation of semen from endangered pheasants: the first step towards a cryobank for endangered avian species

In order to improve the genetic management of bird species within the European Endangered Programs (EEP), a research project on artificial insemination and cryopreservation of Galliformes semen has been developed. The aim of the program is to create a sperm cryobank for threatened bird species. During this study, semen was collected from 17 pheasant species and specific characteristics of ejaculates were analyzed (volume, sperm concentration, motility, pH). Artificial insemination with fresh semen was performed in nine species and with frozen semen in eight species. Inseminations with frozen and thawed semen were made in 17 species. Viability of fresh and frozen semen was assessed in vitro using double stains, eosin and nigrosin. The effect of pH (7-8.5) on viability of fresh and frozen/thawed spermatozoa was also studied. Chicks hatched in eight and three species after insemination with fresh and frozen/thawed semen, respectively. Species varied widely in semen viability: 1-30% of spermatozoa survived freezing and thawing. There was a negative correlation between the viability of frozen spermatozoa and semen pH. In our experimental conditions, the pH of diluents had no effect on semen viability. However, semen with the highest pH had the lowest quality after freezing and thawing. These experiments demonstrated the feasibility of using a very simple and inexpensive method to achieve artificial insemination and cryopreservation of semen in endangered pheasant species.     

Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa

The present study evaluates the effectiveness of the transcervical deep intrauterine insemination (DUI) with a reduced number of frozen-thawed boar spermatozoa in weaned sows. DUI was performed using a specially designed flexible device (length 180 cm, outer diameter 4mm, working channel 1.8mm, working channel's volume 1.5 ml) that was inserted through an artificial insemination spirette to cross the cervix lumen and moved into one uterine horn as far as possible. Spermatozoa diluted in 7.5 ml of BTS were flushed into the uterine horn by a syringe attached to the working channel. In Experiment 1, 111 hormonally treated (eCG/hCG) weaned sows were inseminated once using one of the following three regimens: (1) DUI with frozen-thawed spermatozoa (1000 x 10(6) cells per dose; n=49); (2) DUI with fresh semen (150 x 10(6) cells per dose; n=29, as control of DUI procedure); and (3) cervical insemination with frozen-thawed spermatozoa (6000 x 10(6) cells diluted in 100ml; n=33). No differences (P>0.05) were found for farrowing rates (77.55, 82.76, and 75.76, respectively) or litter sizes (9.31+/-0.41, 9.96+/-0.32, and 9.60+/-0.53 piglets born per litter, respectively) among the groups. In Experiment 2, DUI was performed on the spontaneous estrus in weaned sows (2-6 parity) with 1000 x 10(6) frozen-thawed (40 sows) or 150 x 10(6) fresh spermatozoa (38 sows). The farrowing rate of sows inseminated twice with frozen-thawed spermatozoa (70%) was significantly (P<0.05) lower than with fresh semen (84.21%). No significant difference (P>0.05) was found in litter size between frozen-thawed spermatozoa (9.25+/-0.23 piglets born per litter) and fresh semen (9.88+/-0.21 piglets born per litter). These preliminary results indicate that application of DUI provides acceptable fertility in weaned sows using a relatively low number of frozen-thawed spermatozoa.     

Effect of insemination dose on pregnancy rate in mares

Different insemination doses have been used for artificial insemination(AI) in horses. Since the insemination dose can affect the pregnancy rate, it is important to ensure that an adequate dose be used regardless of the type of inseminationprotocol used. The aim of this study was to find out if it is possible to decrease the insemination dose from 500 x 10(6) progressively motile spermatozoa to 300 x 10(6) progressively motile spermatozoa and still maintain an acceptable pregnancy rate when using extended fresh semen. Thirteen stallions of known fertility and a well-defined group of 64 mares were used in the study. The mares were randomly assigned to 1 of 2 insemination groups. Examination for pregnancy was performed by ultrasonography per rectum approximately 16 d after the last insemination. When using an insemination dose of 300 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 75%. With an insemination dose of 500 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 64%. There was no significant difference in the pregnancy rate between the 2 insemination doses (P = 0.341). We conclude that when using fresh extended semen it is unlikely that an insemination dose of 300 x 10(6) progressively motile spermatozoa would yield a lower pregnancy rate than a dose of 500 x 10(6) progressively motile spermatozoa if stallions with good quality semen are selected.     

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.     

Use of a semen extender containing antibiotic to improve the fertility of a stallion with seminal vesiculitis due to Pseudomonas aeruginosa

A breeding trial was conducted to determine if a semen extender containing polymixin-B sulfate would improve the fertility of a stallion with seminal vesiculitis due to Pseudomonas aeruginosa . Twenty-three mares were bred to the stallion by one of three methods: artificial insemination with raw semen (Group 1, n = 10), artificial insemination with semen mixed 1:1 with a nonfat dry skim milk/glucose extender containing 1000 units/ml polymixin-B sulfate (Group 2, n = 9), or natural service immediately following infusion of the uterus with 100 ml of the same extender (Group 3, n = 4). Artificial breedings contained a minimum insemination dose of 500 x 10(6) progressively motile spermatozoa. All mares were bred every other day while in estrus. Pregnancy status was determined by transrectal ultrasound examination 15 d after the last breeding. First-cycle pregnancy rate for Group 2 mares (78%) was greater (P < 0.01) than for Group 1 mares (10%). There was a tendency (P = 0.10) for the pregnancy rate of Group 3 mares (50%) to be greater than Group 1 mares. The use of a semen extender containing polymixin-B sulfate improved the fertility of this stallion.     

Artificial insemination in domestic cats (Felis catus)

Artificial insemination (AI) in cats represents an important technique for increasing the contribution of genetically valuable individuals in specific populations, whether they be highly pedigreed purebred cats, medically important laboratory cats or endangered non-domestic cats. Semen is collected using electrical stimulation, with an artificial vagina or from intact or excised cauda epididymis. Sperm samples can be used for AI immediately after collection, after temporary storage above 0 degrees C or after cryopreservation. There have been three and five reports on intravaginal and intrauterine insemination, respectively, and one report on tubal insemination with fresh semen. In studies using fresh semen, it was reported that conception rates of 50% or higher were obtained by intravaginal insemination with 10-50x10(6) spermatozoa, while, in another report, the conception rate was 78% after AI with 80x10(6) spermatozoa. After intrauterine insemination, conception rates following deposition of 6.2x10(6) and 8x10(6) spermatozoa were reported to be 50 and 80%, respectively. With tubal insemination, the conception rate was 43% when 4x10(6) spermatozoa were used, showing that the number of spermatozoa required to obtain a satisfactory conception rate was similar to that of cats inseminated directly into the uterus. When frozen semen was used for intravaginal insemination the conception rate was rather low, but intrauterine insemination with 50x10(6) frozen/thawed spermatozoa resulted in a conception rate of 57%. Furthermore, in one report, conception was obtained by intrauterine insemination of frozen epididymal spermatozoa. Overall, there have been few reports on artificial insemination in cats. The results obtained to date show considerable variation, both within and among laboratories depending upon the type and number of spermatozoa used and the site of sperm deposition. Undoubtedly, future studies will identify the major factors required to consistently obtain reliable conception rates, so that AI can become a practical technique for enhancing the production of desirable genotypes, both for laboratory and conservation purposes.     

Effect of site of deposition on the fertility of sheep inseminated with frozen-thawed semen

The widespread use of artificial insemination (AI) in sheep is currently prevented due to the lack of a cost effective insemination technique utilising frozen-thawed semen. The objective of the present study was to determine if the deposition of frozen-thawed semen in the vaginal fornix would result in a pregnancy rate comparable to that achieved following cervical insemination. Multiparous ewes of various breeds were synchronised and inseminated into either the vaginal fornix (n=78) or the cervix (n=79), at 57 h post sponge removal, with frozen-thawed semen. Information on mucus secretion and the depth to which it was possible to penetrate the cervix at insemination (cervically inseminated ewes only) was recorded at the time of AI. Pregnancy rate was subsequently determined either by return to service (oestrus) or after slaughter 30 days post insemination. Insemination site did not significantly influence pregnancy rate using frozen-thawed semen (36.2% compared to 27.6% for cervical and vaginal fornix insemination, respectively; P=0.26). Whilst depth of cervical penetration was positively associated with pregnancy rate (P<0.05), this association needs to be interpreted with caution as none of the ewes where the cervix could not be penetrated (score=0) was pregnant. In conclusion, pregnancy rate following insemination of frozen-thawed semen into the vaginal fornix was within 10% points of that obtained following cervical AI of frozen-thawed semen. As insemination into the vaginal fornix is technically easier than cervical insemination, it may be more practical for use in large scale applications.     

Identification of embryo paternity using polymorphic DNA markers to assess fertilizing capacity of spermatozoa after heterospermic insemination in boars

Differences in sperm fertilizing capacity of males often remain undetected by routine semen parameters. Heterospermic insemination with equal numbers of spermatozoa from 2 males is an accurate method for assessing differences in fertility. Use of heterospermic insemination depends on a reliable, efficient assay to identify paternity of conceptuses or offspring. In this study, polymorphic DNA markers amplified by PCR were tested to determine paternity of Day 5 to 6 embryos. The fertilizing capacity of 2 boars (A and B) with similar semen parameters was compared after homospermic (n=14 gilts) and heterospermic (n=11 gilts) insemination. Single AI's were performed under suboptimal conditions using 1 x 10(9) spermatozoa at 12 to 24 h before ovulation to prompt differences in fertilization and to stimulate sperm competition. The fertilization rate and the number of accessory spermatozoa were determined in Day 5 to 6 embryos. Using 5 different polymorphic DNA markers, paternity could be determined in 95.8% of the embryos. Boar B sired significantly (P<0.05) more offspring than Boar A after insemination with pooled semen, and this was reflected by a significantly (P<0.05) higher number of accessory spermatozoa following homospermic insemination with semen from Boar B, although fertilization rates did not differ between the 2 boars after homospermic insemination. The results suggest that the viability of spermatozoa in the female reproductive tract contributes to differences in fertility rates of males with similar in vitro sperm quality parameters. The number of accessory spermatozoa is a more sensitive measure of boar fertility than the fertilization rate. Polymorphic DNA markers are suitable for verification of parentage even at a very early stage of embryonic development.     

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.     

Time required for spermatozoa to remain in the vagina of the ewe to ensure conception

Oestrous ewes (N = 202) were inseminated with 0.1 ml of semen containing 500 X 10(6) motile spermatozoa and the spermatozoa were flushed from their vagina either immediately or 0.25, 0.5, 1, 2, 4 and 8 h after insemination. Pregnancy was determined by returns to service and laparoscopy. Some ewes became pregnant (10.71%) after spermatozoa had been flushed from the vagina only seconds after insemination and about 40% of ewes became pregnant after spermatozoa had been in the vagina for 15 min. Maximum conception (55%) was achieved when spermatozoa had been in the vagina for at least 2 h. It was concluded that the losses of spermatozoa that occur from the vagina will not influence the chance of a ewe conceiving because sufficient spermatozoa to ensure a normal conception move up the reproductive tract before large losses from the vagina take effect.     

A study of factors affecting the success of human fertilization in vitro. II. Influence of semen quality and oocyte maturity on fertilization and cleavage

The incidence of in vitro fertilization was analyzed with respect to the degree of cumulus dissociation (expansion) at the time of oocyte recovery and also the semen quality. Of the oocytes surrounded by perfectly ("++") or moderately ("+") dissociated cumuli, 78.6% and 30.8%, respectively (P less than 0.001), were fertilized when the husband's semen analysis was in the normal range. The proportion of fertilized oocytes was not decreased in cases of polyzoospermia (greater than 130 X 10(6) spermatozoa/ml), but was decreased (P less than 0.05) when the semen analysis revealed other anomalies: oligozoospermia (less than 15 X 10(6) spermatozoa/ml), asthenozoospermia (less than 50% motile cells) or teratozoospermia (greater than 50% abnormal spermatozoa). The proportion of fertilized eggs cleaving in vitro was unaffected by semen quality but was lower when "+" cumulus oocytes were collected than when "++" cumulus oocytes were obtained (58.3% vs. 87.0%, P less than 0.02). In vitro incubation of the oocyte prior to insemination increased the incidence of fertilization by about 28% for both "+" (22.2 to 50.0%) and "++" (65.7 to 93.9%) cumulus oocytes. Finally, 67.6% of "++" cumulus oocytes developed into embryos when the insemination with spermatozoa from normal semen samples was delayed by several hours, compared with only 29.0% when the conditions were suboptimal ("+" cumulus oocyte, abnormal semen analysis or no delay prior to insemination). Eight pregnancies began following the replacement of 38 embryos in 34 patients. Six spontaneous abortions occurred, and chromosomal abnormalities were proven in the two cases analyzed. Two pregnancies continued for more than 3 months, resulting in term deliveries of two normal babies.     

Evaluation of deep-frozen semen in stallions.

The sperm-rich fraction of stallion semen was collected in an AV and, after dilution in an extender, was cooled to 2--5 degrees C before placing in aluminium tubes for freezing in liquid nitrogen for several hours or months. The spermatozoa in about 200 ejaculates from 36 stallions were examined to compare their survival time, motility and velocity before and after thawing. According to the various indices used, 20% of stallions produced spermatozoa which were unaffected, 60% partly but not seriously affected and the remainder completely inactivated. The velocity of spermatozoa decreased from 51.4 micrometers/sec in the fresh semen to 36.8 micrometers/sec in the thawed semen. The fertilizing capacity of the spermatozoa of frozen--thawed semen of 5 stallions was examined in 14 mares. In all, 65 inseminations were made and the blastocysts were recovered non-surgically from the uterus 7--9 days after ovulation. A 20% drop in blastocyst recovery occurred as the result of freezing and thawing, when the same mares were used for insemination of raw and frozen--thawed semen. The capacity to freeze sucessfully proved to be a specific characteristic of certain stallions. Degenerate blastocysts were not recovered but those resulting from artificial insemination of frozen semen were much smaller in diameter than those following insemination of raw semen.