Artificial insemination of ewes with frozen-thawed semen at a synchronized oestrus. 1. Effect of time of onset of oestrus, ovulation and insemination on fertility

In three experiments, the onset of oestrus, time of ovulation and lambing after intrauterine insemination with frozen-thawed semen were examined following synchronisation of oestrus using intravaginal progestagen-impregnated sponges (inserted for 12 days) and an injection of PMSG at sponge removal.

The number (and percentage) of ewes detected in oestrus 12, 24, 36, 48, 60 and 72 h after sponge removal was 1 (0.3), 2 (0.6), 17 (5.2), 120 (36.7), 65 (20.0) and 10 (3.1) respectively. One hundred and twelve ewes (34.3%) remained unmarked. Egg fertilisation rates were not different between ewes irrespective of time of onset of oestrus or whether or not ewes were marked.

The median time of ovulation with respect to sponge removal (with 95% fiducial limits) for ewes joined with vasectomised rams (10:1) at spronge removal (teased ewes) was 55.8 h (54.61–57.09) and for unteased ewes 59.7 h (58.27–61.12).

In the third experiment, a total of 394 ewes were inseminated by laparoscopy with frozen-thawed semen. The percentage of ewes lambing and lambs born per ewe inseminated, and number of lambs born per ewe lambing for inseminations 48, 60, 72 and 78 h after sponge removal were 45.9, 57.7 and 1.25; 55.1, 72.0 and 1.31; 57.4, 80.9 and 1.41; and 39.3, 60.7 and 1.54, and for 59 control ewes receiving fresh semen by cervical insemination 47.5, 69.5 and 1.46 respectively. The lambing data after insemination with frozen semen was not different to that of the controls. The percentage of ewes lambing and lambs born per ewe inseminated increased with time of insemination at 48, 60 and 72 h (linear, P < 0.01) but was lower for inseminations at 78 h after sponge removal. Number of lambs born per ewe lambing increased with time of insemination after sponge removal (linear, P < 0.05).     

Insemination time and dilution rate of cooled and chilled ram semen affects fertility

Adult Merino ewes (n=448) were apportioned into two groups and inseminated with: extended at 30 degrees C with skim milk and stored for 6h at 15 degrees C (cooled semen) or extended with skim milk-citrate trisodium with egg yolk and stored for 24h at 5 degrees C (chilled semen). Each group was further subdivided according to the time of cervical insemination at 42, 46 and 50h after pessary (MAP-60 mg) removal and according to the dilution of the semen (120 x 10(6) spermatozoa in 0.05, 0.1 and 0.2 ml). The pregnancy rate after insemination with cooled semen was 50% better than that after chilled semen (56.7 vs. 37.5%; P<0.001). Pregnancy rate was not affected by the volume of insemination; however, there was a tendency of increased lambing rate with an insemination dose of 0.1 cc (1:2, dilution), especially when the ewes were inseminated with cooled semen. The effect of time on insemination was significant only in ewes inseminated with chilled semen at 5 degrees C (P<0.01). Insemination carried out 46 h after pessary removal resulted in higher pregnancy and lambing rate (36.5, 31.1; 52.0, 45.3; and 24.0, 20.0 at 42, 46 and 50h, respectively). Pregnancy of ewes inseminated with chilled semen at 46 h after pessary removal was similar to that obtained using cooled semen (52.0 vs. 56.7%). From this study, it is concluded that advancing the time of insemination with chilled semen at 5 degrees C improves pregnancy and that the lambing obtained under these conditions is similar to the one obtained with cooled semen.     

Fertility of fresh and frozen rabbit semen inseminated at different times is indicative of male differences in capacitation time

Some reports indicate that sperm from different males differ in capacitation time, and other reports suggest that freezing sperm may affect their capacitation time. These two variables were specifically studied in rabbits in a fertility trial with 96 does inseminated with approximately 1.6 million motile fresh or frozen sperm from three different bucks at 15, 10, 5, and 0 h before expected ovulation. Fresh semen averaged 84% live (unstained) sperm and 88% had normal acrosomes; corresponding values for frozen sperm were 44% and 54%. On the basis of does that became pregnant, average litter size with fresh semen was 5.5 and with frozen semen was 4.8 (p greater than 0.05), but overall, does bred with frozen semen produced fewer young (p less than 0.05). On the basis of total does and total semen, average litter size from insemination at 15, 10, 5, and 0 h was 2.8, 4.2, 3.8, and 1.7, and average litter size for the three bucks was 4.0, 1.8, and 3.6. There was no interaction of type of semen (fresh or frozen) with the other variables in the model (p greater than 0.05). Bucks and time of insemination affected both the proportion of does that were pregnant and litter size (p less than 0.01). A major interaction between buck and time of insemination (p less than 0.01) was due apparently to both differential sperm survival and probable capacitation time among bucks. This major interaction should be considered in designing in vitro and in vivo fertility studies, and for selecting males for use in artificial insemination.     

The effects of different insemination regimes on fertility in mares

This study investigated the effects of different artificial insemination (AI) regimes on the pregnancy rate in mares inseminated with either cooled or frozen-thawed semen. In essence, the influence of three different factors on fertility was examined; namely the number of inseminations per oestrus, the time interval between inseminations within an oestrus, and the proximity of insemination to ovulation. In the first experiment, 401 warmblood mares were inseminated one to three times in an oestrus with either cooled (500 x 10(6) progressively motile spermatozoa, stored at +5 degrees C for 2-4 h) or frozen-thawed (800 x 10(6) spermatozoa, of which > or =35% were progressively motile post-thaw) semen from fertile Hanoverian stallions, beginning -24, -12, 0, 12, 24 or 36 h after human chorionic gonadotrophin (hCG) administration. Mares were injected intravenously with 1500 IU hCG when they were in oestrus and had a pre-ovulatory follicle > or =40mm in diameter. Experiment 2 was a retrospective analysis of the breeding records of 2,637 mares inseminated in a total of 5,305 oestrous cycles during the 1999 breeding season. In Experiment 1, follicle development was monitored by transrectal ultrasonographic examination of the ovaries every 12 h until ovulation, and pregnancy detection was performed sonographically 16-18 days after ovulation. In Experiment 2, insemination data were analysed with respect to the number of live foals registered the following year. In Experiment 1, ovulation occurred within 48 h of hCG administration in 97.5% (391/401) of mares and the interval between hCG treatment and ovulation was significantly shorter in the second half of the breeding season (May-July) than in the first (March-April, P< or =0.05). Mares inseminated with cooled stallion semen once during an oestrus had pregnancy rates comparable to those attained in mares inseminated on two (48/85, 56.5%) or three (20/28, 71.4%) occasions at 24 h intervals, as long as insemination was performed between 24 h before and 12 h after ovulation (78/140, 55.7%). Similarly, a single frozen-thawed semen insemination between 12 h before (31/75, 41.3%) and 12 h after (24/48, 50%) ovulation produced similar pregnancy rates to those attained when mares were inseminated either two (31/62, 50%) or three (3/9, 33.3%) times at 24 h intervals.In the retrospective study (Experiment 2), mares inseminated with cooled semen only once per cycle had significantly lower per cycle foaling rates (507/1622, 31.2%) than mares inseminated two (791/1905, 41.5%), three (464/1064, 43.6%) or > or =4 times (314/714, 43.9%) in an oestrus (P< or =0.001). In addition, there was a tendency for per cycle foaling rates to increase when mares were inseminated daily (619/1374, 45.5%) rather than every other day (836/2004, 42.1%, P = 0.054) until ovulation.It is concluded that under conditions of frequent veterinary examination, a single insemination per cycle produces pregnancy rates as good as multiple insemination, as long as it is performed between 24 h before and 12 h after AI for cooled semen, or 12 h before and 12 h after AI for frozen-thawed semen. If frequent scanning is not possible, fertility appears to be optimised by repeating AI on a daily basis.     

Influence of sperm number and seminal plasma on fertility of progestagen-treated sheep in confinement

Progestagen-impregnated vaginal sponges + PMSG were used to synchronize oestrus in crossbred adult ewes which were inseminated 56 h after sponge removal with 0.5 ml diluted semen containing 400, 200, 100, 50 or 25 x 10(6) spermatozoa per insemination. The diluent was skim milk-citrate or pooled seminal plasma. There was no difference in reproductive performance due to the insemination medium. Fertility (no. of ewes lambing) after insemination of 400 or 200 x 10(6) spermatozoa was 68% and was similar to that observed after natural service at progestagen-induced oestrus. When less than or equal to 100 x 10(6) spermatozoa were inseminated, fertility fell markedly and the number of lambs per ewe inseminated decreased. A decrease in litter size also occurred. The data indicate that insemination of 200 x 10(6) spermatozoa, i.e. less than 10% of the number in a single ram ejaculate, allows normal conception rates in progestagen-treated ewes.     

Selected times of insemination with microencapsulated bovine spermatozoa affect pregnancy rates of synchronized heifers

This experiment was designed to test whether spermatozoa encapsulated in an alginate poly-L-lysine matrix had an extended fertile life in vivo after insemination. Estrus was synchronized in 417 primiparous Friesian and Jersey heifers with a system based on a CIDR-B intravaginal device before the heifers were inseminated either during proestrus (24 h after device removal) or at estrus (48 h after device removal). Pregnancy rates to first inseminations did not differ between the 24 and 48 h inseminations (61 vs 60.6%) with liquid semen diluted in Caprogen (control) but differed with encapsulated semen (45.1 vs 68.6%). The difference in pregnancy rates between the 2 types of semen was more pronounced (P < 0.08) in the animals that were visually detected in estrus. The mean survival time of spermatozoa in the female reproductive tract following insemination at the 24-h insemination time was estimated to be 50 +/- 7.5 h. The increased pregnancy rate with insemination of encapsulated spermatozoa at 48 h could have been due to this process predisposing spermatozoa to capacitate soon after insemination.     

Fertility of fresh and frozen-thawed goat semen during the nonbreeding season

A total of 4109 does of a local Greek breed (Capra prisca) were synchronized with intravaginal MPA-sponges and PMSG, and 24 bucks of Alpine (n = 8), Saanen (n = 8) and Damascus (n = 8) breeds were used for studying the fertility of nonfrozen and frozen-thawed semen during the nonbreeding season (June to August). Artificial insemination (AI) was performed once (50 to 55 h after sponge withdrawal) or twice (36 and 60 h after sponge withdrawals with fresh semen (collected during the nonbreeding season, stored at 16 degrees C and inseminated within 6 h) or frozen semen (prepared from the same bucks during the preceding breeding season). The induction of estrus was successful, varying between 91.0 and 95.0%. The form of semen (fresh or frozen-thawed used for inseminating the synchronized does affected their fertility: the overall kidding rate with fresh semen (65.5%) was higher (P < 0.05) than that with frozen-thawed semen (53.4%). The fertility level was also affected by the number of inseminations performed: the overall kidding rate was significantly higher (P < 0.001) in the does inseminated twice with fresh or frozen-thawed semen (70.4 and 59. 1%, respectively) than in those inseminated only once (48.9 and 44.9%, respectively). Finally, the breed of the buck used for preparing the fresh or the frozen-thawed semen affected the fertility level of the does. The kidding rate was higher in does inseminated with fresh semen prepared from bucks of the Damascus breed than from bucks of Saanen or Alpine breed. However, when frozen-thawed semen was used the kidding rate was lower in does inseminated with semen prepared from bucks of the Damascus breed than from bucks of the Alpine or Saanen breed. It is concluded that the fresh semen of Alpine, Saanen and Damascus breed bucks, born and raised under the climate conditions prevailing in Greece (34 degrees to 41 degrees N), can be used successfully during the nonbreeding season (June to August) for inseminating does.     

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).     

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%.     

Artificial insemination of ewes with frozen-thawed semen at a synchronised oestrus. 2. Effect of dose of spermatozoa and site of intrauterine insemination on fertility

Three experiments were conducted to examine the effect of dose of inseminate, number of uterine horns inseminated and site of insemination on subsequent fertility of Merino ewes after synchronisation of oestrus, with progestagen-impregnated sponges (inserted for 12 days) and an injection of PMSG, and intrauterine insemination with frozen-thawed semen.The percentages of ewes lambing after insemination with 0.5, 5, 25 and 50 × 10⁶ spermatozoa were 29.3, 26.8, 56.3 and 62.1% respectively. A similar trend was observed in a second test resulting in 23.5, 38.8 and 53.1% ewes lambing after insemination with 5, 10 and 20 × 10⁶ spermatozoa respectively.The percentage of ewes lambing was higher for ewes inseminated in two uterine horns than one horn (76.8 vs. 44.9, P < 0.001). When semen was deposited in the tip, middle and bottom of the uterine horn, the percentages of ewes lambing and lambs born per ewe inseminated were 43.6 and 52.7, 52.8 and 84.9, and 41.2 and 64.7% respectively. Although site of insemination did not affect the percentage of ewes lambing, the percentage of lambs born per ewe inseminated was higher after insemination in the middle of the uterine horn than at the other sites (P < 0.001).     

Microscopic and flow cytometric semen assessment of Dutch AI-bucks: effect of semen processing procedures and their correlation to fertility

This study was done to determine the effects of processing techniques on the quality of semen from Dutch AI-bucks with the view on improving pregnancy rates after artificial insemination (AI) with liquid or frozen-thawed semen. Motility of spermatozoa was estimated under a microscope whereas the percentage live spermatozoa and the percentage live spermatozoa with intact acrosomes were determined by means of flow cytometry. Aspects of semen processing that were investigated are storage temperature of liquid semen (i), the effect of glycerol on liquid-stored semen (ii), removal of seminal plasma (iii) and type of extender (iv). The correlation between semen quality and fertility rates in inseminated does was also investigated. The percentage motile spermatozoa in semen stored in liquid form for 72 h progressively declined over time, irrespective of whether storage occurred at 4 or 18 degrees C. The percentage motile spermatozoa in semen stored at 18 degrees C was similar to that in semen stored at 4 degrees C if stored for 24 h but lower if stored for 48 h. Goats differ in the sensitivity of their spermatozoa to the deleterious effects of glycerol. Neither the removal of seminal plasma nor the type of extender had any effect on semen quality before freezing but semen frozen in a Tris-citric acid-glucose (TCG) buffer with egg yolk without removal of the seminal plasma had better quality after thawing than semen frozen in another diluent or after removal of seminal plasma. Remarkably no significant correlation between fertility and membrane integrity of spermatozoa could be found. Thus, although integrity assays for spermatozoa are useful to asses resistance to semen handling, the validity of these assays for predicting fertility is questioned.     

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.     

Intrauterine insemination enhances fertility of frozen semen in superovulated ewes

Superovulated ewes were inseminated with fresh or frozen semen in a factorial experiment which compared two techniques of artificial insemination; i.e. conventional cervical deposition and intrauterine deposition at laparoscopy. Similar fertilization rates resulted from insemination with fresh semen at cervical (81% of ova from 11/11 ewes) and intrauterine (83% of ova from 10/12 ewes) sites. These results approached those observed in a naturally-mated group (95% of ova from 5/5 ewes). In ewes inseminated with frozen semen, fertilization rate was markedly reduced (P less than 0.05) after cervical insemination (11% of ova from 3/11 ewes) and partly restored (P less than 0.05) after intrauterine insemination (50% of ova from 8/11 ewes).     

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 °C and 15 °C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 °C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 °C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 μL semen at a sperm concentration of 20 × 10⁶/mL. Semen was stored at 5 °C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 °C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 °C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P < 0.001) up to 72 h after semen collection.     

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.     

Reproductive response of ewes synchronized with different lengths of MGA treatments in intrauterine insemination program

A total of 415 fat tailed ewes were randomly assigned to two groups to assess the effect of duration of melengestrol acetate (MGA) (9 versus 12d) administration on reproductive parameters associated with laparoscopic artificial insemination. At the end of MGA treatment, ewes in each group were subdivided and inseminated with one of two different insemination doses (10×10(7) or 20×10(7) sperm per 0.5 ml insemination dose) of fresh diluted semen. Inseminations were carried out 11-18 h after first detected estrus. Ewes were screened for their return to oestrus from 10 to 21 days post AI and inseminated at their returned oestrus. Pregnancy diagnosis was done from approximately 55 days after insemination in both synchronized and return estrus. For short (9-day) and long (12-day) term MGA treated groups, estrus rates were 62% versus 89% (P<0.0001), respectively. Ewes (n=115) that returned to estrus were inseminated (7-11h after estrus detection) with fresh diluted semen at different doses (20×10(7) or 40×10(7) or 60×10(7) sperm per 0.5 ml insemination dose). Pregnancy rates were 41% and 44% for short term and long term MGA treated ewes, respectively. Pregnancy rate of ewes which returned to oestrus was 53.4%. There was a significant (P<0.05) increase in pregnancy rates (38-52% for 11-16 h; 63% for 17-18 h) when insemination was held at 17-18 h after first detected estrus following MGA treatments. Pregnancy rates were found to be similar in ewes inseminated with 10×10(7) (36%) or 20×10(7) (47%) motile spermatozoa at first AI, and 20×10(7) (44%) or 40×10(7) (59%) or 60×10(7)(48%) at second AI. It was concluded that short term MGA treated ewes were recorded with lower estrus rates but was similar to pregnancy rates with long term MGA treatment. Acceptable pregnancy rates were achieved in MGA induced estrus when insemination is conducted at 17-18 h after estrus onset and with 20×10(7) sperm per 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.     

Effect of sterile service on estrus duration, fertility and prolificacy in artificially inseminated dairy goats

The effect of sterile service on estrus duration, fertility and prolificacy in artificially inseminated dairy goats during breeding season was studied. Nubian does (n=126) were divided into 2 equal groups: service and control. Estrus was synchronized with intravaginal sponges containing either fluorgestone acetate (FGA; 40 mg) or medroxiprogesterone acetate (MAP; 60 mg) for 12 or 14 d, respectively. Two vasectomized teaser bucks were used to detect estrus at 6-h intervals for 5 d after sponge removal (0600, 1200, 1800 and 2400 h). The teasers were fitted with aprons and permitted to mount all does in both groups, but to penetrate only the service does within the first 12 h of estrus. Does in both groups were inseminated twice at 12 and 24 h after estrus was first detected, using 1 straw per insemination containing 200 million of cooled spermatozoa from 1 buck. The semen was placed in mid-cervix. Estrus duration for the service and control does was (mean +/- SD) 29.4 +/- 6.5 and 41.8 +/- 9.6 h, respectively. Fertility for the service does was 73.7% (46/63); for control does it was 58.7% (37/63). Prolificacy was 2.1 (96/46) and 2.0 (74/37) for service and control does, respectively. Estrus duration (P<0.001) and fertility (P<0.05) differed between the service and control group, but prolificacy was similar (P>0.05). It is concluded that sterile service reduces the duration of estrus and increases fertility in artificially inseminated dairy goats.     

Intravaginal insemination of bitches with fresh and frozen-thawed semen with addition of prostatic fluid: use of an infusion pipette and the Osiris catheter

One hundred fifty-two bitches of seven breeds were vaginally inseminated with fresh or frozen-thawed semen of 10 stud dogs of respective breeds. The semen was supplemented with prostatic fluid before insemination. In experiment 1 bitches of each breed were randomly assigned to three treatment groups, consisting of 29 females (group 1), 33 females (group 2) and 32 females (group 3). In group 1 bitches were inseminated into vagina with fresh semen using a bovine infusion pipette. In group 2 bitches were inseminated into vagina with fresh semen using the Osiris catheter. In group 3 bitches were inseminated with frozen-thawed semen with the Osiris catheter. The number of sperms in each insemination dose was adjusted to 300 x 10(6). In experiment two bitches were randomly assigned to two treatment groups, consisting of 30 females (group A) and 28 females (group B). In group A bitches were inseminated with fresh semen, whereas in group B with frozen-thawed semen. Osiris catheter was used in both groups. The total number of sperms was adjusted to provide 250 x 10(6) of progressively motile spermatozoa in each insemination dose. In experiment 1 the pregnancy rates/whelping rates were 86.2/82.8%, 81.8/81.8% and 59.4/59.4% for groups 1, 2 and 3, respectively. The differences between group 1 and 3 were statistically significant (p < 0.05). The litter sizes at birth/litter sizes at weaning were 5.8+/-2.3/5.4+/-2.0, 6.3+/-1.4/5.7+/-1.0 and 3.9+/-1.2/3.5+/-1.5 in groups 1, 2 and 3, respectively. The litter size at birth and at weaning was reduced (p < 0.05) when frozen-thawed semen was used for insemination (group 3). There were not significant (p > 0.05) differences in the litter size between groups 1 and 2. In experiment 2 pregnancy rates/whelping rates and litter sizes at birth/litter sizes at weaning were 86.7/86.7%, 60.7/57.1% (p < 0.05) and 6.1+/-1.6/5.7+/-1.7, 4.0+/-1.4/3.8+/-1.4 (p < 0.05) in groups A and B, respectively. This study shows that results of AI with a fresh semen using a bovine infusion pipette and the Osiris catheter are equivalent. The results of the use of the Osiris catheter for vaginal insemination of frozen-thawed dog semen extended with prostatic fluid after thawing are not encouraging. The pregnancy rate, whelping rate and litter size are reduced when frozen-thawed, prostatic fluid-supplemented semen is vaginally deposited using the Osiris catheter.     

Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen

In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 degrees C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. Group 3 mares were inseminated once with 2 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. All mares were given 2500 IU i.v. hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered i.m. 10 mg of PGF2 alpha and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 x 10(9) progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 x 10(6) motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 degrees C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 x 10(9) progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 x 10(9) progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 x 10(9) progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 x 10(9) progressively motile spermatozoa given on two consecutive days. However, a shorter interval (< or = 24 h rather than > 0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.