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.     

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.     

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 degrees C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 degrees C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.     

In vitro and in vivo fertility of ram semen cryopreserved in different extenders

Seminal traits of frozen-thawed (FT) ram semen and in vitro and field fertility in native Portuguese breeds were evaluated in 4 experiments. In exp. 1 and 2 the cryopreservation capacity of 2 extenders, E1 (15% egg yolk-EY) and E2 (4.5% EY and trehalose) was compared through morphological evaluation and in vitro fertilizability of FT ram semen. Exp. 3 aimed to determine the usefulness of in vitro homologous/heterologous fertilization tests as tools for predicting ram fertility. Exp. 4 was conducted to verify if the identified differences between the 2 extenders could be confirmed by field fertility. E1 showed a better cryoprotective action expressed by higher in vitro and field fertility results. In conclusion, EY is difficult to be replaced in ram semen extenders. Heterologous fertilization seems to be a useful tool for predicting fertility of FT ram semen.     

Estrus synchronisation and fertility in gilts using a synthetic progestagen (Allyl Trenbolone) and inseminated with fresh stored or frozen semen

An orally active synthetic progestagen was administered at two dosage levels to synchronise estrus in gilts. Fertility following insemination with either fresh stored or frozen semen was determined by examining surgically recovered ova for cleavage, and numbers of spermatozoa attached to the zona pellucida, or enumeration of embryos in gilts slaughtered 30 days post insemination. There was no significant difference (P>0.05) between treated and control groups in the duration of estrus or in fertility as determined by cleavage of ova. A significantly (P<0.001) shorter interval to estrus and better synchronisation was obtained with both treatment groups than with the control group. The mean interval from the end of treatment to the onset of estrus for the untreated controls and the treated groups receiving 12.5 and 15 mg compound per day was 11.25 +/- 10.4 SD; 5.6 +/- 0.52 SD and 7.3 +/- 5.3 SD. Fresh semen yielded significantly (P<0.01) more cleaved ova than frozen semen.     

Artificial insemination of sheep: the fertility of semen extended in diluents containing egg yolk and inseminated soon after dilution or stored at 5 degrees C for 24 or 48 hours.

In an experiment involving the artificial insemination (AI) of 1175 ewes, ram semen was diluted 10- or 30-fold in a buffered glucosesaline solution containing either 1.5% or 6% (v/v) egg yolk. Part of each semen collection was used undiluted for control AI of 10⁸ sperm/dose. Diluted samples were reconcentrated to 10⁹ sperm/ml by centrifugation and, from these preparations, 10⁸ spermatozoa were inseminated in a standard volume of 100 μl. Fertility was assessed by 28–45 day non-returns to oestrus.The processes of dilution and reconcentration caused a significant drop in the non-return rate (NRR) and cooling to 5°C and storage for up to 48 hrs at this temperature gave a further large, and highly significant, reduction in NRR. There was no significant effect of level of egg yolk in the diluent on NRR.     

Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen

Precise data on fertility results following peri- and postovulatory insemination in spontaneously ovulating gilts is lacking. Using transcutaneous sonography every 4 h during estrus as a tool for diagnosis of ovulation, the effects of different time intervals of insemination relative to ovulation were investigated with liquid semen (Experiment 1, n=76 gilts) and frozen semen (Experiment 2, n=80 gilts). In Experiment 3 (n=24 gilts) the number of Day-28 embryos related to the various intervals between insemination and ovulation was determined after the use of liquid semen. Using liquid semen the fertilization rates based on Day-2 to Day-5 embryos and the number of accessory spermatozoa decreased significantly in gilts inseminated with 2 x 10(9) spermatozoa per dosage in intervals of more than 12 h before or more than 4 h after ovulation. In the time interval 4 to 0 h before ovulation, comparable fertilization rates were obtained using frozen semen (88.1%) and liquid semen (92.5%). Fertilization rates and numbers of accessory spermatozoa decreased significantly when gilts were inseminated with frozen semen more than 4 h before or 0 to 4 h after the detection of ovulation. The percentage of Day-28 embryos was significantly higher following preovulatory insemination compared to inseminations 0 to 4 h and 4 to 8 h after ovulation. It is concluded that the optimal time of insemination using liquid semen is 12 to 0 h before ovulation, and 4 to 0 h before ovulation using frozen semen. The results stress the importance of further research on sperm transport and ovulation stimulating mechanisms, as well as studies on the time of ovulation relative to estrus-weaning intervals and estrus duration.     

Effect of cervical and vaginal insemination with liquid semen stored at room temperature on fertility of goats

The effect of vaginal and cervical deposition of liquid semen stored at room temperature on the fertility of goats was tested in a field trial in which 217 Norwegian Dairy goats aged between 6 months and 7.5 years from 14 farms were inseminated after natural oestrous. Cervical insemination with 200 x 10(6) spermatozoa resulted in 25-day non-return and kidding rates of 87.0 and 78.0%, and vaginal insemination gave 85.5 and 74.3%, respectively. There was no significant difference between the cervical and vaginal inseminations (P = 0.59 for the 25-day non-return and P = 0.40 for the kidding rates). Farm had a significant effect on the 25-day non-return rate (P = 0.03) but not on the kidding rate (P = 0.07). There were no significant differences between the fertility rates for different bucks (P = 0.36 for the 25-day non-return and P = 0.15 for the kidding rates). Fertility results after vaginal insemination were encouragingly high. Vaginal insemination is a simple, less costly and time consuming technique compared to others, also bringing into focus the animal welfare aspects of the artificial insemination procedure. As the final goal is to establish a technique that could be applied similarly on a large scale by all farmers, vaginal insemination must be considered as a method that would simplify the use of liquid buck semen in Norway.     

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.     

Effect of different equilibration times and extenders on deep freezing of buffalo semen

Thirty semen collections from 3 Murrah buffalo bulls were frozen in Tris yolk glycerol (TY-G) and Citric acid whey glycerol (CAW-G) extenders using 2, 4 and 6 hours equilibration times and 7 percent glycerol level. Sperm motility after freezing was studied at an interval of 15 minutes 7 days and 30 days storage in liquid nitrogen. Sperm survivability was found to be better at all the stages of deep-freezing using 4 hours equilibration time. Significant differences (P 0.01 ) were observed between extenders and equilibration times.     

Effects of heating the testes and epididymides of rams by scrotal insulation on fertility and embryonic mortality in ewes inseminated with frozen semen.

Fertilization rate and embryonic mortality were assessed in 636 ewes inseminated in each uterine horn with 50 x 10(6) frozen spermatozoa from four control rams and from four rams submitted to a moderate (1.4-2.2 degrees C), but repeated, intermittent (16 h/day for 21 consecutive days) increase in their subcutaneous scrotal temperature by means of scrotal insulation. Pregnancy was assessed twice in each ewe from concentration of progesterone in blood plasma at 17 days and by ultrasound at 65 days after insemination. No differences were observed in the pregnancy rate at 17 days between ewes inseminated with semen collected from control rams (56.0, 65.2, 66.7 and 60.3%) and from heated rams (60.6, 71.8, 63.6 and 48.2%) before or after 4, 15 and 21 days of heating, respectively. In contrast, the rate of embryonic mortality between 17 and 65 days after insemination was significantly higher at days 4, 15 and 21 in the heated rams (78.7, 78.6 and 93%) than in the control rams (55, 59 and 65.7%). These results indicate that an intermittent slight, but repeated, increase in the subcutaneous scrotal temperature could induce a significant increase in the embryonic mortality rate. As these changes were apparent on day 4 of heating, an effect must have occurred on sperm stored in the epididymis.     

Effect of antioxidant supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa

The objective of this study was to evaluate quality of chilled dog semen processed with extenders containing various antioxidants. Single ejaculates from five dogs were always pooled and evaluated for concentration, sperm motility, progressive motility (RSF-movement), viability, acrosomal integrity and by the hypo-osmotic swelling (HOS)-test. Also, superoxide (O(2)(-)) production, hydroxyl radicals (OH) and total reactive oxygen species (tROS) were determined. Pooled semen was divided in seven aliquots (for control and test conditions), which were diluted to a final concentration of 67x10(6)spermatozoa/ml with TRIS-glucose-egg yolk extender with or without the following supplements: control (without antioxidants), vitamin C (0.5mM), N-acetyl-l-cysteine (NAC; 0.5mM), taurine (0.2mM), catalase (100u/ml), vitamin E (0.1mM) and 5-(4-dimethylamino-phenyl)-2-phenyl-penta-2,4-dienoic acid (B16; 0.1mM). The semen aliquots were chilled and preserved at 4 degrees C. Portions of chilled semen were removed at 24 and 72h, and semen quality was evaluated after rewarming. At 24h the mean (+/-S.E.M.) sperm motility was higher (p<0.001) when vitamin E, taurine and B16 were added in the extender, whereas more spermatozoa with RSF-movement were observed (p<0.001) in the vitamin E, catalase, B16 and taurine groups. Sperm viability was higher (p=0.040) in B16 and vitamin E groups and the percentage of swollen spermatozoa was higher (p=0.002) only in the B16 group. Acrosomal integrity and OH were not significantly influenced by any of the antioxidants tested. Superoxide production was significantly lower when vitamin C, B16 and vitamin E were added in semen extenders compared with the control (p=0.017). All antioxidant groups, except vitamin C and NAC, contained less tROS compared to the control group, but only the B16 group value differed significantly (p=0.05). At 72h sperm motility was higher (p<0.001) when vitamin E, catalase, B16, taurine and NAC were added in the extender. More spermatozoa with RSF-movement were observed (p<0.001) in the vitamin E, catalase, B16, taurine and NAC treatment groups. Sperm viability was higher (p=0.001) when vitamin E, B16, taurine and vitamin C were added in semen extenders. HOS-test percentages were higher (p=0.016) in the B16, vitamin E, catalase and NAC groups. Acrosomal integrity was not influenced in any case. Production of O(2)(-) was significantly higher using catalase compared to all the other groups (p=0.006), while OH was not significantly influenced by any of the antioxidants tested. The addition of vitamin E, catalase and B16 in semen extenders resulted in significantly lower tROS values compared with the controls (p<0.0005). The results suggest that vitamin E and B16 had the most pronounced effect in preserving semen quality of chilled dog spermatozoa.     

Effect of different diluents on goat semen fertility

Skim milk (SM) is considered to be the most widely employed extender for goat sperm used for artificial insemination (AI). However, the fertilizing life span of sperm stored in milk or milk-based extenders does not exceed 12h. Besides some seminal plasma components, such as a protein fraction from the goat bulbourethral gland secretion (SBUIII), interacts with some milk fractions and inhibits the spermatozoa motility. The aim of this study was to prolong the survival of buck semen and its fertility. Buck ejaculates were diluted to a final concentration of 100x10(6)spermatozoa/ml with three different diluents: SM, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPOL+hyaluronic acid (TEMPOL+HA). At 7h from dilution 42 goats were inseminated with semen diluted with SM (short-term semen) while after storage for 24h, 44 and 45 goats were inseminated with semen diluted with TEMPOL and TEMPOL+HA (long-term storage), respectively. At day 50 from AI the percentages of pregnant goats were 71.4% (30/42) with SM, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, with significant differences between SM and TEMPOL+HA. The kidding rate was 66.7% (28/42) with SM diluent, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, without significant differences among treatment groups. In conclusion, it is possible to maintain good fertility in goats after AI with semen stored for 24h in TEMPOL.     

Effect of eCG on the pregnancy rate of ewes transcervically inseminated with frozen-thawed semen outside the breeding season

An experiment was conducted to determine whether factors affecting pregnancy rate out-of-season are associated more with transcervical artificial insemination (T-AI) procedures or with the reproductive state of the ewe. Twenty Finncross ewes were treated with progesterone sponges, and at sponge removal (0 h) 10 ewes were treated with eCG. Blood samples were collected for LH and progesterone analyses, and follicular development was monitored using ultrasonography. Ewes were inseminated from 48 to 52 h with 200 million motile frozen-thawed spermatozoa. The incidence of estrus, LH surges and ovulation was greater (P < 0.01) and intervals to these responses were shorter (P < 0.01) in the eCG-treated ewes. The number of follicles > 5 mm was higher (P < 0.05) in eCG-treated than control ewes. Progesterone concentrations increased and remained elevated through Day 19 in 7 eCG-treated and in 1 control ewe, and these ewes were pregnant based upon ultrasonographic examination. The results demonstrate that the T-AI technique using frozen-thawed semen produces a relatively high (70%) pregnancy rate out-of-season. The pregnancy rate was found to reflect primarily the reproductive condition of the ewe.     

Artificial insemination of sheep: the effect of semen diluents containing egg yolk on the fertility of ram semen.

Results from an artificial insemination (AI) experiment revealing the effect of semen dilutents containing egg yolk on the fertility of ram semen are presented. Ram semen was diluted 30-fold in buffered glucose-saline solution containing . 375, 1.5, or 6% v/v egg yolk and a portion of each was used soon for the AI of ewes or was incubated at 35 degrees C for 1 hour prior to AI. Some of the semen collection was used undiluted for AI of 10(8) spermatozoa/dose. All diluted samples were reconcentrated by centrifugation so that each dose was 10(8) spermatozoa in a volume of 100 mcl. 1146 ewes were inseminated. Fertility was assessed from 28 to 45 day nonreturns to estrus and nonreturn rates (NRRs) were expressed as percentages for the various treatments. Undiluted semen (controls) revealed 69%, semen used soon after dilution, .375% yolk in dilutent 58%, 1.5% yolk 50%, 6% yolk 42%; diluted semen incubated for 1 hour before use, .375% yolk 49%, 1.5% yolk 51%, and 6% yolk 39%. NRR was significantly depressed by dilution (p less than .001) and by increasing amounts of egg yolk (p less than .01) in the dilutent. Incubation of diluted semen before AI caused a small fall in NRR.     

Freezability, enzyme leakage and fertility of buffalo spermatozoa in relation to the quality of semen ejaculates and extenders

Forty-eight semen ejaculates from four Surti buffalo bulls were studied under split sample technique to establish the effects of initial semen quality and tris fructose yolk glycerol (TFYF), egg yolk citrate glycerol (EYCG) and lactose yolk glycerol (LYG) extenders on the freezability, fertility (based on 3412 AI) and extracellular release of spermatozoal enzymes pre and postfreezing. The overall mean activity of GOT, GPT, AKP, ACP and LDH enzymes in the postthaw seminal plasma increased significantly (P<0.01) above prefreeze levels, whereas sperm motility decreased. Freezability and the release of these enzymes were not influenced by the types of extenders used except for AKP release, which was significantly (P<0.01) lower in TFYG diluent. The pre and postfreeze sperm motility was significantly higher (P<0.01) and the leakage of GOT, AKP and ACP was lower in 36 semen samples with an initial motility above 70% than in the 12 samples in which initial motility was between 60 and 70%. The effects of interactions between motility groups, diluents and freezing periods were statistically nonsignificant for both freezability and leakage of all five enzymes. Fertility rate of frozen semen produced in TFYG diluent was significantly (P<0.05) higher (42.69%) than in the other diluents and was followed by that of EYCG (39.78%) and LYG (37.50%) with an overall mean of 40%. Sperm post-thaw motility had significantly (P<0.01) negative correlations with the release of enzymes: GOT (-0.948); GPT (-0.859); AKP (-0.673); ACP (-0.951) and LDH (-0.764). Fertility rates showed high negative correlations with the release of all five enzymes. Freezability was positively correlated with fertility (+0.405). Significant positive correlations were also observed for the release of GOT with that of GPT (+0.944); AKP (+0.574); ACP (+0.911) and LDH (+0.839): GPT with ACP (+0.795) and LDH (+0.870): and ACP with AKP (+0.725) and LDH (+0.577). These findings stressed the use of simple estimates of GOT and AKP leakage as markers for the assessment of freezability and fertility, and also the importance of initial good quality semen and the suitablity of extenders (TFYG) in the production of frozen buffalo bull semen for better fertility rates.     

Effects of synthetic GnRH on litter size in ranch mink bred once or twice

Female ranch mink of the Pearl variety were injected with saline (100 μl), GnRH (2 μg) or HCG (50 IU) and mated 7, 8 or 9 days later. Mean number of young in litter (± SEM) for saline treated females was 1.4 ± 0.87; GnRH 4.8 ± 0.55 and HCG 3.4 ± 0.9. A fourth group were injected with GnRH immediately after each of two matings and resultant mean litter size in this group was 5.7 ± 0.44. The results suggest that GnRH can effectively replace the first mating in ranch mink and that GnRH after each of two matings may enhance preovulatory development and/or ovulation.