Survival and fertility of ram spermatozoa frozen in pellets, straws and minitubes

The post-thaw survival and fertility of ram spermatozoa frozen in pellets, 0.25- and 0.5-ml PVC straws, and 0.25-ml minitubes were examined. In 5 experiments, a freezing height of 6 cm above the level of liquid nitrogen was optimal for 0.25- and 0.5-ml straws, whereas 4 cm was best for the 0.25-ml minitubes. Post-thaw motility of spermatozoa was lower for semen frozen in straws and minitubes than in pellets (Experiment 1: 43.7 vs 53.4%, P < 0.001), but after freezing was better in 0.5-ml straws and 0.25-ml minitubes than in 0.25-ml straws (Experiment 1: 44.9 vs 41.3%, P < 0.05; Experiment 2: 49.6 vs 46.8%, P < 0.01). Sperm motility was also better for 1:8 (semen:diluent) pre-freezing dilution rate (50.5%) than for 1:4 (45.6%, P < 0.01) and 1:2 (39.8%, P < 0.001) but not the 1:16 (49.5%) dilution rate. Dry ice was a better freezing medium than liquid nitrogen vapor (49.2 vs 46.9% motile spermatozoa, P < 0.001). The post-thaw motility of spermatozoa was similar for the three freezing packages if the semen was loaded at 5 degrees C, but motility was poorer for semen loaded into 0.25-ml straws than 0.25-ml minitubes at 30 degrees C (P < 0.05). In a fertility test, pregnancy rates were influenced by rams (3 rams, P < 0.05) and freezing package (pellets vs 0.25-ml minitube vs 0.25-ml straw vs 0.5-ml straw, P < 0.05) but not freezing medium (liquid nitrogen vapor vs dry ice). More ewes were pregnant after insemination with pellet-frozen semen (106/150, 71%) than with semen frozen in 0.25-ml straws (85/150, 57%; P < 0.05) and in 0.5-ml straws (83/150, 55%; P < 0.01) but not minitubes (98/150, 65%). It was concluded that minitubes provide a useful alternative to pellets as a storage package for ram spermatozoa, allowing for individual dose identification and easier storage while maintaining a fertility rate indistinguishable from that obtained with pellet-frozen semen.     

Therapeutic donor insemination with frozen semen.

Although it is now accepted that cryopreserved semen must, on ethical and medicolegal grounds, be used for donor insemination many clinicians still believe that it has an unacceptably reduced fecundability rate as compared with fresh semen. We studied the outcome of 81 recipients who started therapeutic donor insemination (TDI) treatment during 1986 in a program that used exclusively cryopreserved semen; 55 had never undergone TDI and were receiving the first series (six cycles), 6 were receiving the second series (also six cycles), and 20 had achieved pregnancy through TDI previously and were starting the treatment again. Insemination with semen stored in 0.5-ml French straws was performed daily during the periovulatory period while the modified Insler score was 10 or greater out of 15. A total of 42 (52%) of the recipients became pregnant within six TDI cycles; 4 (10%) had a spontaneous abortion. An average of 4.8 straws were used per cycle among those who became pregnant and 5.1 per cycle among those who did not. On average 2.6 cycles were required to achieve pregnancy. The overall fecundability rate was 14.6%. We conclude that a TDI program involving exclusively frozen semen can be operated with a success rate comparable to rates achieved with fresh semen if a simple, established cryopreservation method and an uncomplicated clinical management protocol are used.     

A comparison of three packaging techniques using two extenders for the cryopreservation of canine semen

The progressive motility of frozen-thawed canine semen was used as a criterion to compare methods of semen cryopreservation. Twenty-one ejaculates from 7 dogs were frozen in 2 extenders, Tris-citrate (TC) and BES-lactose (BL), in each of 3 packaging techniques (pellets, 0.5-ml, and 2.5-ml straws). Duplicate samples were frozen on dry ice (pellets) or in liquid nitrogen vapor (straws). Least squares means for the percentage of post-thaw progressive motility (PTPM) were greater for TC (33.2 ± 1.7) than for BL (20.9 ± 1.7; P<0.0001). Freezing in pellets (PTPM = 34 ± 2.3) resulted in greater PTPM than freezing in either 0.5-ml (24.7 ± 1.6) or 2.5-ml (22.1 ± 2.3) straws (P<0.001). The percentage of PTPM of spermatozoa frozen in TC pellets was greater than that in TC 2.5-ml straws or in BL in any packaging method (P<0.05). The percentage of PTPM of spermatozoa from semen extended in BL was greater in pellets than in 0.5 or 2.5-ml straws (P<0.05).     

Insemination Results with Slow-Cooled Stallion Semen Stored for approximately 40 Hours

Semen from 3 stallions was extended using 2 methods (Kenney extender and a modified Kenney extender), slowly cooled, and stored for 41 ± 6 (s.d.) h before insemination. An insemination dose (40 ml) contained 1.5-2 billion spermatozoa. In the experiment, 26 mares were inseminated in 30 cycles. The pregnancy rate per cycle obtained with sperm stored in the Kenney extender was 87% (n=15). When the semen was extended with the modified extender, centrifuged and stored, the pregnancy rate was 60% (n=15). Inseminations were done every other day until ovulation was detected. If a mare ovulated more than 24 h after the last insemination, she was inseminated also after ovulation. The single-cycle pregnancy rate was 58% when the mares were inseminated only before ovulation (n=19) but the rate was 100% when the inseminations were done both before and after ovulation (n=9) or only after ovulation (n=2). The difference in pregnancy rates was significant (p<0.05), indicating that postovula-tory inseminations probably serve to ensure the pregnancies. The extending and handling methods used in this study resulted in a combined pregnancy rate of 73%, and appear thus to be useful for storing stallion semen for approximately 2 days.     

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.     

French field results (1985-2005) on factors affecting fertility of frozen stallion semen

Results on procedures for freezing stallion semen and the subsequent fertility during 20 years are presented. The present system applied in French National Stud includes: (1) a freezing protocol (dilution in milk, centrifugation and addition of freezing extender (INRA82+egg yolk (2%, v/v)+glycerol (2.5%, v/v) at 22 degrees C, a moderate cooling rate to 4 degrees C and freezing at -60 degrees C/min in 0.5-ml straws); (2) selection of ejaculates showing post-thaw rapid motility >35%; and (3) an insemination protocol (mares examined once daily, two AI of 400 x 10(6) spermatozoa 24 h apart before ovulation, sufficient number of straws to have the possibility to perform six AI of 400 x 10(6) total spermatozoa, i.e. 2.4 x 10(9) total spermatozoa available per mare per season). This system was applied to >110 stallions per year, the average post-thaw motility of ejaculates was 50% (>1800 ejaculates) before selection. The semen freezability was defined as the number of selected ejaculates divided by the total number of ejaculates frozen. Of the stallions, 5, 4, 5, 21 and 64% had semen freezability of 0-10, 10-33, 33-60, 60-90 and over 90%, respectively. Per-cycle pregnancy rate was 45-48% (>1500 mares per year, 1.8 cycles per mare) and foaling rate 64%. In comparison, per-cycle pregnancy rate and foaling rate of mares hand-mated to stallions were 57-59% and 64%, respectively. The average number of straws used was 32-35 (1.75 x 10(9) total spermatozoa) per mare per season. According to our results and the literature, the most important factors for improving fertility of frozen equine semen include: (1) a low concentration of glycerol (2-3.5% final concentration); (2) a suitable base extender for freezing like Lactose-Glucose EDTA or INRA82; (3) a post-thaw motility >30-35%; and (4) a sufficient number of spermatozoa per mare per season (1.5-2 x 10(9) total spermatozoa for two to three cycles) divided into small units. Numbers of spermatozoa, lower than 750.10(6) total spermatozoa per cycle, could result in lower per-cycle pregnancy rate with higher additional costs for management of mares. Because there are no particular regulations on quality and quantity of equine semen in the European Community, there is a need for the uniformity of information about frozen semen. A codification is suggested, based on the number of spermatozoa available per mare per season, the post-thaw motility and the final glycerol concentration.     

Pregnancy rates following AI with sexed semen in Mediterranean Italian buffalo heifers (Bubalus bubalis)

The use of sexed semen in farm animal production and genetic improvement has been shown to be feasible with variable degree of efficiency in a number of species, and proved to be economically viable in cattle. In the last two decades, various newly developed reproductive technologies applicable in buffaloes have mushroomed. Recently, following the birth of the first buffalo calves using AI with sexed semen, commercial interest to exploit sexing of semen in this species too is aroused. In order to verify the successful adoption of this technology in the buffalo, the present study on the use of sexed semen for AI was carried out and compared with conventional artificial insemination using nonsexed semen. A total of 379 buffalo heifers were used for synchronization of ovulation using the Presynch protocol in the South of Italy. Selected animals at the time of AI were randomly allocated to three different experiment groups: (1) 102 animals subjected to AI in the body of the uterus with sexed semen (SS body); (2) 104 animals subjected to AI in the horn of the uterus with sexed semen (SS horn); and (3) 106 animals subjected to AI in the body of the uterus with conventional nonsexed semen (NSS body). Semen of three buffalo bulls was sexed by a collaborating company and commercially distributed in 0.25 mL straws with a total of 2 million sexed spermatozoa. Pregnancy rates were first assessed at Day 28 following AI, and rechecked at Day 45 by ultrasound. Pregnancy rates were nonsignificantly different between animals inseminated with sexed or nonsexed semen: 80/206 (38.8%) and 40/106 (37.7%), respectively (P = 0.85). However, site of insemination of sexed semen affected pregnancy rate significantly as higher pregnancy rates were obtained when sexed semen was deposited into the body rather than the horn of the uterus: 46/101 (45.5%) and 34/105 (32.3%), respectively (P = 0.05). In conclusion, the use of sexed semen in buffalo heifers gave satisfactory and similar pregnancy rates when compared with conventional nonsexed semen. Deposition of sexed semen into the body of the uterus, however, increased pregnancy rates significantly.     

Fertility of frozen-thawed stallion semen extended in lactose-EDTA-egg yolk extender and packaged in 1.0-ml straws

The fertility of frozen-thawed and fresh semen from each of three stallions was compared in an experiment with a randomized block design using 128 mares. Semen was collected every third day, extended in lactose-EDTA-egg yolk extender at a concentration of 500 × 10⁶ progressively motile sperm per 1.0 ml, and frozen in individual-dose, 1.0-ml straws (1.9 mm × 267 mm). The same stallions were collected daily for inseminations with fresh semen. For each insemination dose with fresh semen, 300 × 10⁶ progressively motile sperm were added to 10 ml of heated skim milk extender. Mares were inseminated daily from the second day of estrus through the end of estrus. Of 52 ejaculates processed and frozen, 38% were discarded because < 35% of the sperm were progressively motile after thawing. Based on rectal palpations on day 50 post-ovulation, pregnancy rates for inseminations during one estrus to semen from the three stallions were 17, 33 and 35% for frozen-thawed semen and 60, 62 and 64% for fresh semen. Pregnancy rates with frozen semen from two of the three stallions were 54% of the rates attained with fresh semen.     

Ultrastructural and cytochemical characterization of follicular cell types in bovine (Bos taurus) cumulus-oocyte complexes aspirated from small and medium antral follicles during the estrus cycle

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.     

Comparative viability of bovine sperm frozen on a cryomicroscope or in straws

The accuracy and repeatability of freezing rates and effects of evaporation were examined using a new cryomicroscope system to establish its usefulness in assessing the development of cryopreservation protocols for bovine semen. Post-thaw sperm plasma membrane integrity, as assessed by using combinations of fluorescent stains and flow cytometry, was used in evaluating protocols for freezing spermatozoa on the cryomicroscope. Semen was diluted in Test-yolk (20%) extender containing 7% glycerol and frozen in 0.5-ml straws, 0.25-ml straws (over liquid nitrogen for 8 min) or in a quartz crucible using a Linkam BCS 196 cryomicroscope. Thawed samples were diluted with Hepes buffered medium containing 0.1% bovine serum albumin (BSA) and stained with either carboxymethylfluorescein diacetate (CMFDA) or SYBR-14 each in combination with propidium iodide (PI). Flow cytometry analysis of the samples revealed 2 major populations: 1) spermatozoa with intense green fluorescence (stained with CMFDA or SYBR-14), which were classified as plasma membrane-intact and 2) spermatozoa with intense red fluorescence, (stained with PI), which were classified as plasma membrane-damaged. Samples frozen using the cryomicroscope contained 29 and 26 % plasma membrane-intact (PMI) sperm cells, as assessed by CMFDA and SYBR-14, respectively. Cryopreservation of spermatozoa in 0.5-ml straws resulted in 22 and 20% plasma membrane- intact sperm cells, while spermatozoa frozen in 0.25-ml straws resulted in 34 and 31% PMI sperm cells for CMFDA and SYBR-14, respectively. No significant difference was observed (P > 0.05) for PMI spermatozoa stained with either CMFDA or SYBR-14. In addition, the ability to recover spermatozoa after freezing on the cryomicroscope establishes the Linkam BCS 196 as a useful tool for the study of sperm cell cryopreservation.     

Ovulating induction methods in rabbit does: the pituitary and ovarian responses

The aim of this study was to compare the pituitary and ovarian responses in rabbit does subjected to different methods of ovulation induction. Forty-eight receptive females were randomly distributed into six groups (N = 8) and were inseminated with standard glass catheters. Buserelin intramuscular (BM) does were inseminated using a pool of fresh heterospermic semen and an intramuscular injection of 1 μg of buserelin acetate to induce ovulation. Buserelin intravaginal (BV) does were inseminated in a similar way, but ovulation was induced with the GnRH analogue (10 μg of buserelin acetate) combined with 0.5 mL of semen extender. The raw semen (R) and saline groups (S) were inseminated with undiluted semen or saline, respectively, without any inducer of ovulation. Another group (A) received lumbar anaesthesia (1.5 mL of 2% lidocaine), and only the empty catheter was introduced into the vagina. The AR does were treated the same way as group A but were inseminated with raw semen instead of an empty catheter. Blood samples were collected to determine the LH concentrations before and after AI (30, 60, 90, and 120 minutes). Ovulation, pregnancy, and conception rates were determined after euthanasia on day 14 post AI. Ovulating does had higher mean LH concentrations than nonovulating does (197.9 vs. 45.9 ng/mL; P < 0.05). The ovulation rates of buserelin intramuscular and intravaginal does were 100%, and the pregnancy rates were 87.5% and 100%, respectively. Rabbit does in groups A and AR did not ovulate and had similar mean plasma LH concentrations after 60 minutes compared with the S group (49.4 and 49.2 ng/mL vs. 41.6 ng/mL, respectively), which reached ovulation and pregnancy rates of 37.5%. Does inseminated only with raw semen had an ovulation rate of 75% and a pregnancy rate of 62.5%; they also demonstrated higher plasma LH concentrations than does of the S, A, and AR groups. In conclusion, ovulation in rabbit does can be induced by exogenous GnRH administration (im and intravaginal). The high plasma LH concentration and ovulation rate in the R group with respect to the S and A groups could weakly indicate the presence of some molecules in the seminal plasma that could act on or be absorbed by vaginal mucosa. Sensory stimulation and “seminal factors” probably exert a synergy on the ovulation response as demonstrated by the comparison of LH release and the ovulation response in the R, S, RA, and A groups.     

Comparison of different extenders for the cryopreservation of Atlantic salmon spermatozoa

Fifteen extenders were produced by adding dimethyl sulfoxide (DMSO) at 8, 10 or 12% of diluent volume to 5 diluents. All extenders were cooled to 4 degrees C. Pooled Atlantic salmon (Salmo salar ) semen with greater than 90% progressive motility was kept at 4 degrees C and added to each extender so that the semen was diluted 1:3 (semen:extender). The equilibration time was less than 5 minutes at 4 degrees C. The extended semen was loaded into 0.5-ml straws and was cooled from 4 degrees C to -90 degrees C at a rate of 30 degrees C per minute. The straws were then plunged into liquid nitrogen for storage. Fluorometry was used to determine the viability of the semen in each of the extenders after freezing and thawing. Cryopreservation of Atlantic salmon semen in Extender 3 (0.137 M NaCl, 0.011 M KCl, 0.004 M Na(2)HPO(4).7H(2)O, 7.5 g/l L-alpha-lecithin and 12% dimethyl sulfoxide) and Extender 12 (0.100 M KHCO(3), 0.0065 M reduced glutathione, 0.125 M sucrose and 12% dimethyl sulfoxide) resulted in significantly (P<0.05) lower percentages of dead spermatozoa than for the other extenders. Furthermore, there was a significantly (P<0.05) lower percentage of dead cells in Extender 3 than in Extender 12.     

Effect of Timing of Frozen Semen Insemination on Pregnancy Rate in Mares

Thirty-four mares were inseminated with frozen semen from one stallion during 2 oestrous cycles, every 48 h until ovulation took place and within 12 h after ovulation. Semen was frozen using the Colorado method. The insemination dose was from 200 to 400×106 progressively motile spermatozoa. Ovaries were examined every 12 h to determine time of ovulation. Examination for pregnancy was carried out using ultrasonography, 15 days after ovulation. Thirty-five per cent of mares inseminated < 24 h and 23% of mares inseminated between 24 - 48 h before ovulation were pregnant (p = 0.388). The pregnancy rate in all mares inseminated before ovulation was 30%. In the mares inseminated within 12 h of ovulation, it was 18% (p = 0.253). Younger mares (aged 4-10 yr) had a higher pregnancy rate (59%) than older mares (aged 11-15 yr) (23%), but the difference was not statistically significant (p = 0.057).     

Fertility results after different thawing procedures for ram semen frozen in minitubes and mini straws

The effect of different thawing procedures for ram semen frozen in minitubes and mini straws on the fertility of sheep was tested in a field trial in which 727 Norwegian crossbred ewes, aged between six months and five-and-a-half years from nine farms, were inseminated with frozen-thawed semen in natural estrous. Minitubes were thawed at 70 degrees C for 8 s (T70) and mini straws either at 70 degrees C for 5 s (S70), 50 degrees C for 9 s (S50), or 35 degrees C for 12 s (S35). Cervical insemination with 200 x 10(6) spermatozoa resulted in 25-day non-return rates of 78.7, 69.0, 73.6, and 72.9% (overall 73.6%), respectively, and lambing rates of 77.6, 66.1, 71.4, and 68.9% (overall 71.0%), respectively. There was a significantly higher lambing rate for T70 compared to S35 (P=0.03) and S70 (P=0.02), respectively, but not compared to S50 (P=0.29). Age of the ewes (P=0.02), farmers (P=0.02) and the interaction between farmer x straw type/thawing temperature (P=0.01) had a significant effect on the lambing rate. In conclusion, the superior fertility results achieved for minitubes compared to mini straws have to be carefully evaluated in relation to the possible application of a more rational semen production and simplified semen handling at AI, when using mini straws thawed at 35 degrees C.     

Effect of insemination timing on the fertilizing capacity of frozen/thawed equine spermatozoa

A breeding trial was conducted to evaluate the effect of insemination timing on the fertility of mares bred with frozen/thawed equine semen. One stallion and 60 reproductively sound, estrous-synchronized mares were included in the study. Mares were assigned to one of three groups (n = 20): 1) insemination with fresh semen every other day during estrus from detection of a 35-mm follicle until ovulation, 2) insemination with frozen/thawed semen every day during estrus from detection of a 35-mm follicle until ovulation or 3) insemination with frozen/thawed semen once, within 6 h after ovulation. Single-cycle 18-d pregnancy rates resulting from insemination with fresh semen (70%), preovulation insemination with frozen/thawed semen (60%) and postovulation insemination with frozen/thawed semen (55%) were not different (P > 0.05). Possibly, equivalent pregnancy rates could be achieved with frozen/thawed semen using either daily inseminations until ovulation occurs or frequent ovarian palpations with a single post-ovulation insemination. Further studies regarding the effect of insemination timing on stallion fertility are needed since the present investigation included only one stallion and a small number of mares.     

Cryopreservation of seabream (Sparus aurata) spermatozoa

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.     

Feasibility of using hard gelatin capsules for the packaging and deep-freezing of bull semen

Various packaging systems have been used for deep freezing of semen. In this study, feasibility of using hard gelatin capsules was established. Of the four types of capsules developed and tested, polymer-treated capsules were found to be suitable for the purpose, and were therefore used subsequently. French medium (0.5 ml) straws were used for control. Five semen samples from each of 12 bulls were processed and included for study. Semen was frozen by fast-freezing. Parameters studied after thawing of semen were comparable for the two methods. Upon analysis, the percentages of progressive motile spermatozoa, live spermatozoa and morphologically abnormal spermatozoa obtained for semen frozen in hard gelatin capsules and French medium straws were found to be nonsignificant. The percentage of intact acrosomes was found to be significantly higher (P < 0.05) for semen frozen - thawed in straws as compared to semen in capsules.     

Successful pregnancies in cows following double freezing of a large volume of semen

The objective of the following paper is to describe a new technology for large volume and double freezing of semen in 12 mL test tubes. Semen from two different bulls was frozen with a new technique using 12 mL test tubes and was refrozen after thawing in mini straws. All freezing was done in a "Multi thermal gradient" (MTG) freezing apparatus, which moves the container at a constant velocity (V) through a thermal gradient (G) producing a controlled cooling rate B = (G) x (V). Each of the two bulls ejaculated were evaluated for post thaw motility in the lab and then in a field trial which was carried out in a split sample mode. We inseminated 105 cows after a double freezing/thawing cycle, and another 123 cows were inseminated with semen frozen in mini-straws and a conventional method. The results showed a 75 +/- 5% post thaw motility after freezing a 12 mL test tube and 50 +/- 5% after a second freezing/thawing in mini-straws, respectively. Controlled vapour freezing showed a 60 +/- 10% post thaw motility. The results of the field trial showed a pregnancy rate of 44% (47/105) for the double freezing group in comparison to 45.5% (56/123) for the controlled group. These results can be beneficial for large volume freezing, and therefore for bull semen cryobanking in a large volume which will be followed by second freezing in a regular insemination volume.     

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.