Large batch freezing of bull semen: effect of time of freezing and fructose on fertility.

Large-scale batch freezing of bull semen should be done in a processing schedule that yields the highest fertility and when it can be fitted efficiently into the work schedule. Conflicting reports have appeared on survival and fertility of bull sperm frozen within a few hours of semen collection or on the next day. To study this problem, a factorially arranged experiment with semen from 10 bulls was conducted, comparing whole milk-glycerol semen extender with and without fructose, and semen frozen in 0.5-ml straws after 4 versus 18 h of equilibration at 5 degrees C. Both fructose and 18 h of equilibration resulted in a small but significant improvement in freeze-thaw survival of sperm. A field trial followed with replicated semen collections from nine bulls processed in a whole milk-glycerol control extender frozen after 4 h of equilibration versus the addition of 1.25% (wt/vol) fructose to whole milk glycerol divided to freeze sperm after 4 and 28 h of equilibration. Semen from these bulls was used to inseminate 14,775 first-service cows. The 56-d nonreturn rates obtained for these three treatments were 74.7, 74.3, and 73.9%, respectively. As there was no difference in fertility, it would appear that programs to freeze sperm in whole milk extenders the same day of collection or the day after semen collection should yield equivalent results.

Within-herd use of boar semen at 5 degrees C, with a note on electronic monitoring of oestrus.

A system was designed to allow a small swine farm in a northern latitude to use its own boars for artificial insemination (AI) conveniently. Semen was collected twice weekly for 3 day use (days 0, 1 and 2), extended in an egg yolk extender and stored at 5 degrees C. Farm personnel were trained to manage the entire AI programme. For simplicity all semen collected was used for insemination. In the first test 47 gilts and 15 sows were inseminated with semen from four boars. One boar was subfertile with a farrowing rate of 36%. The averages for the other boars ranged from 71 to 100%. Then semen was collected from seven boars and all was used to inseminate 70 gilts and 55 sows with 3 x 10(9) or more sperm. Overall 63% farrowed an average of 10.1 piglets per litter. Litter size for sows was 1.5 piglets larger than for gilts. There was no difference in farrowing rate when more than 3 x 10(9) sperm were inseminated. The feasibility of initiating a complete AI programme within a small herd using herd boars was established. However, selection of the boars, use of only high quality semen, and experience with detecting oestrus was required to increase the farrowing rate. The use of various agents to protect sperm against cold shock below 15 degrees C is worthy of further investigation. A new type of electronic probe, which measures the conductivity of cervical mucus, could be helpful if a boar is not available for conventional detection of oestrus.

Sperm output and hormone concentrations in Finn and Dorset rams exposed to long- and short-day lighting.

Seasonal changes in photoperiod have a substantial effect on sexual behavior and reproduction in rams. Little information is available on sperm output from high libido versus average libido rams subjected to intensive semen collection while being exposed to controlled short versus long photoperiods. Six Finn and six Dorset rams were compared in a reversal design, which allowed rams of both breeds to be exposed to 8 h versus 16 h of light. During each of two 84-d periods rams were subjected twice to an initial depletion of epididymal sperm reserves by collecting up to 26 ejaculates of semen in 3 d, followed by up to 10 ejaculates per day, 1, 3, 5, and 7 d after the initial depletion. A total of 2673 semen samples were collected. Nearly twice as many ejaculates (63.6% of the total) were obtained from Finn rams as from Dorset rams during both the initial and subsequent 3-d sperm depletion periods. This difference in libido was associated with obtaining 33.6 +/- 3.1 x 10(9) sperm from Finn rams versus 10.0 +/- 2.2 x 10(9) sperm from Dorset rams during the initial depletion period (P<0.05). Changes in photoperiod did not affect sperm output (P>0.05) in Finn rams, but may have affected Dorset rams. With 16 h of light, prolactin was significantly (P<0.05) increased in both breeds, particularly in Finn rams. Testosterone in both breeds followed an endogenous rhythm, not affected by the change in controlled photoperiods.

Inhibition of sperm motility does not affect live-dead separation of bull sperm by glass beads.

AIM: This study was designed to explore factors which influence binding of dead versus live sperm to glass filters. METHODS: Multiple semen collections from bulls were used to explore selective filtration of bull sperm as influenced by nonlethal inhibition of sperm motility with fluoride, killing of sperm by quick-freezing, alteration of the glass surface with silicone, and different intervals of sexual rest between semen collections. RESULTS: A comparison of glass spheres 100, 200 and 390 microm in diameter indicated that 200 microm spheres were optimal for selective filtration. Quantitative separation of live from dead sperm was demonstrated with a correlation between the percentage of motile sperm and retention of sperm by the filter of r = -0.87 (P < 0.05). Up to 0.02 mol/L NaFl did not alter the proportion of sperm retained by the filter despite inhibiting sperm motility during filtration, an inhibition which was reversible. Proportions of live-dead sperm, based upon eosin staining, were unaffected by fluoride. Coating the glass spheres with silicone greatly reduced selective filtration. Dead sperm adherence to glass was reduced and resistance to NaFl inhibition was increased by daily ejaculation versus one-week intervals of sexual rest. CONCLUSION: These studies indicate that the adherence of sperm to glass is primarily due to some form of physico-chemical change accompanying death of the sperm cell independent of active sperm motility. This attraction between the sperm plasma membrane and glass is modified by the age of the ejaculated sperm. This information is useful in evaluating different clinical procedures used for sperm separation.

Accessory sperm as an indication of fertilizing ability of rabbit spermatozoa frozen in egg yolk-acetamide with detergent.

Many factors besides initial semen quality affect fertilization rates as sperm interact with the environment of the female reproductive tract. One of these factors is sperm transport, which can be evaluated by accessory sperm counts. Dutch rabbits were used to test the effects on sperm transport, fertilization, and production of young when sodium and triethanolamine lauryl sulfate (STLS) detergent was added to a medium for sperm cryopreservation. When STLS was added in 10 concentrations ranging from 0% to 2.0% (vol/vol) to an egg yolk-acetamide semen extender, optimal post-thaw motility of rabbit sperm occurred when 0.2% to 0.7% STLS was included. However, when 0%, 0.2%, and 0.7% STLS was included to cryopreserve sperm used for insemination, the fertilization rates were 95%, 68%, and 75%, and the corresponding mean numbers of accessory sperm per embryo were 13.1, 1.7, and 0.4 (P < .05). In another experiment, increasing the acetamide concentration from 0.75 M to 1.25 M decreased fertilization rates from 66% to 35%, and was associated with 4.5 and 0.6 accessory sperm per embryo (P < .05). In the final experiment, 48 does inseminated with sperm cryopreserved with 0%, 0.35%, and 0.70% STLS were allowed to produce young. Corresponding pregnancy rates were 56%, 56%, and 31% (P < .05), and litter sizes were 5.6, 4.1, and 4.2 (P > .05). In these studies, low concentrations of STLS improved motility of frozen-thawed sperm, but fertilization and pregnancy rates were reduced. Sperm transport was correspondingly reduced, and the accessory sperm count provided a reliable measure of the effect of STLS on fertility in contrast to the assessment of the percentage of motile sperm.

Blood catalase and haematocrit values in a breeding colony of Dutch-belted rabbits.

Rabbit seminal plasma catalase is much higher than in the semen of other mammals, and differences appear to be inherited. Because of the scarcity of information on rabbit blood catalase and haematocrit in Dutch-belted rabbits, an investigation of possible effects of gender, age and genetics on these variables was undertaken. There were 191 rabbits sampled at 2-3 months, 130 at 12 months and 61 at 18-24 months of age. There was no age effect on the haematocrit values and on blood catalase activity. At 12 months of age males had an average haematocrit value of 44% compared with 40% for females (P < 0.05). Corresponding average catalase values were 431 and 356 units/ml of blood (P < 0.05). Also catalase was measured in the semen and blood of 34 males, and males differed in both their blood and semen catalase activity (P < 0.05). The correlation between the two traits was r = 0.44. Heritability (h2) estimates, based on 231 rabbits were 0.40 for blood catalase activity, and 0.26 for haematocrit. The genetic correlation between the two variables was 0.83 (P < 0.05). These studies are consistent with the literature in that female rabbits have a slightly lower haematocrit value than males, and this is associated with a lower catalase activity. This appears to be the first report of a study that compares rabbit blood catalase in males and females of different ages. Preliminary evidence that differences may have a heritable basis is consistent with previous studies on rabbit semen catalase.

Freezability of spermatozoa from Finn and Dorset rams in multiple semen extenders.

Ten semen extenders were tested in two experiments for cryopreservation of semen collected from four Finn and four Dorset rams. Two ejaculates of semen were combined from each ram for testing each extender treatment. The extenders consisted of a series of commonly used egg yolk-TRIS media with and without sodium and triethanolamine lauryl sulfate (STLS), a similar extender with 3-N-morpholino propane sulfonic acid (MOPS), and milk and whey extenders. In Experiment 1, extender treatments were replicated with three sets of collections from the eight rams, and in Experiment 2 with two sets. The egg yolk-TRIS-glycerol-STLS (EY(1)TSTLS) extender was significantly superior to other extenders except whole milk in protecting the sperm during freezing and thawing. In Experiment 1, a 20% egg yolk-TRIS-glycerol-STLS extender preserved 71% of the progressively motile Finn sperm (post-thaw divided by pre-freeze percentage of motile sperm), and 76% of the Dorset sperm. In Experiment 2, the corresponding values for the same EY(1)TSTLS extender used with Finn and Dorset sperm were 86 and 64%, respectively. Without STLS the egg yolk extenders were significantly less effective in protecting cryopreserved ram sperm. This egg yolk-TRIS extender, containing STLS and glycerol, may hold promise for freezing ram sperm that could be used successfully for intracervical insemination.

The rabbit as a model for reproductive and developmental toxicity studies.

The rabbit has many advantages as a nonrodent and second model for assessing the effects of toxic agents on semen quality, fertility, developmental toxicity, and teratology. The male and female reproductive systems of the rabbit are described, and data on growth, sexual development and reproduction are compared with mice, rats, and humans. Techniques for semen collection and evaluation in the male, and artificial insemination, superovulation, embryo culture, and embryo transfer in the female are included as useful procedures in toxicity testing. Examples of the use of rabbits and experimental replication for toxicity testing are given. Special features of the visceral yolk sac and development of the chorioallantoic placenta of the rabbit are compared with rodents. The rabbit extraembryonic membranes more closely resemble the human than do the rodents, in some respects. The use of the rabbit in developmental toxicity and teratology studies is discussed.

High catalase content of rabbit semen appears to be inherited.

Reactive oxygen species, as hydrogen peroxide, can be damaging to sperm. Most species have little protective catalase in their semen, but rabbit semen contains substantial amounts of catalase. The objective of the present study was to characterize a substantial number of Dutch rabbits for seminal content of catalase and determine whether differences were inherited. Usually, 4 or more semen samples were analyzed per male. Catalase was measured by a gasometric procedure. In Experiment 1, the correlation between duplicate determinations was r = .99, and between 2 sets of semen samples from 55 males it was r = .95. There was a significant difference (P < .05) among pairs of males from 6 litters. In Experiment 2, semen from each of 11 males collected at an interval of 1 year contained an average of 13 and 12 units of catalase per mL of semen in consecutive years. The correlation between pairs of samples was r = .85. This indicated that the condition was permanent, and possibly genetically controlled. Experiment 3 analyzed the catalase content of semen from 7 sires and 32 sons. The heritability of seminal catalase concentration was 0.48. These studies indicate that rabbit seminal plasma is high in catalase, and that a substantial portion of the differences among males are under genetic control.

Importance of inseminating only cows in estrus.

Reproductive efficiency is a key component in successful dairy farm management. A study was initiated to evaluate the incidence and consequences of inseminating dairy cows in the middle of the estrous cycle or while pregnant. In a research herd of 242 Holsteins, managed for reproduction under typical farm conditions, milk progesterone (P4) was assayed 3 times per wk for at least 120 d postpartum. The P4 cycle was compared with the estrus detection and breeding records and pregnancy diagnosis 6 wk after insemination. About 19% of the inseminations were performed when P4 was high in the estrous cycle and in pregnant cows. Insemination of pregnant cows led to an estimated 17% induced embryonic death or abortion. In Israel, inseminators are extensively trained to detect cows not in estrus. They reject about 16% of the cows submitted for reinsemination, with a 95% accuracy of rejection of 44% of the cows that were pregnant. The pattern of submission of cows for reinsemination in areas around New York State was similar to Israel and to the experimental herd. These results indicate that more careful submission and rejection can reduce the unnecessary use of semen, reduce abortions and minimize long calving intervals, all contributing to the success of a dairy herd operation.

Embryo survival, uterine fluids and tubal SEM in progesterone-asynchronized rabbits.

Survival of embryos exposed to several concentrations of uterine proteins and changes in tubal morphology in rabbits given low preovulatory doses of progesterone (P4) that had previously not affected ovulation or fertilization, but caused severe embryo mortality, were studied. In experiment 1, 332 morulae were cultured for 24 h in a control medium containing < 0.5 to > 3.0 mg x mL(-1) of Day 3 uterine fluid proteins. There was no difference in blastocyst development nor implantation to Day 12 following transfer of the blastocysts to recipients, except fewer implants developed in the BSA control. In experiment 2 the oviducts and uteri of control and P4-treated does were examined by SEM for 8 days following ovulation. Secretory cells in the oviducts and to a lesser extent in the uteri were stimulated by P4 treatment for 3 to 4 days after ovulation. Morphology of ciliated cells was unaffected. The subtle changes did not fully account for P4-induced embryo mortality in vivo.

Fertility of rabbit sperm exposed in vitro to cadmium and lead.

The heavy metals Cd2+ and Pb2+ have been associated with male reproductive toxicology, including possible inhibition of sperm undergoing hyperactivated motility, indicative of capacitation. The objective of the present study was to test fertility of rabbit sperm exposed to Cd2+ or Pb2+ in vitro, followed by insemination of superovulated does. Semen was washed to remove seminal plasma and minimize possible binding of the heavy metals by proteins. Only 400,000 treated or control sperm were inseminated as a sensitive test of treatment, and the time sperm resided in the female before possible fertilization was varied by inseminating from 0 to 12 h after ovulating the does. Only 6 of 22 males tested showed appreciable spontaneous hyperactivation and neither Cd2+ nor Pb2+ affected hyperactivation, or presumably associated capacitation. Sperm from four of these six males were used to inseminate 66 rabbits, and 1483 oocytes and embryos were collected about 27 h later. No effect of 0.1 mM Cd2+ on fertilizing ability of sperm was found (75% fertilization with control sperm and 78% with treated sperm). With 0.025 mM Pb2+ the fertilization rate in pregnant does only was 82% for controls and lower (68%) with treated sperm. These tested concentrations of Cd2+ and Pb2+ are much higher than reported concentrations in semen of exposed workers.

Short communication: an electronic probe versus milk progesterone as aids for reproductive management of small dairy herds.

A simple probe especially designed to take electrical resistance measurements at different positions in the anterior vagina of a cow was compared with milk progesterone determinations on 108 cows. Milk samples were taken 3 x weekly, 21 to 60 d postpartum, at the time of insemination, and 21 to 23 d later. Electrical resistance measurements were made on a similar schedule. In 10 other herds, 187 cows had only milk samples taken. No cows with high milk progesterone values became pregnant when inseminated, but the electrical resistance values were less accurate in designating which cows were suitable or unsuitable for insemination. Both low milk progesterone and low electrical resistance values 21 to 23 d after insemination provided an early and accurate indication of a need for reinsemination. These indicators were consistent with 94 to 100% of these cows being diagnosed as not pregnant 6 to 8 wk later. Daily probing, starting about 19 d after a previous insemination, could serve as an early check of pregnancy and assist in identifying cows for immediate reinsemination.

Cadmium affects testes and semen of rabbits exposed before and after puberty.

Semen quality and testicular characteristics were measured in 92 rabbits in three controlled experiments with males exposed to cadmium chloride (Cd) when 12 or 27 weeks old. Doses of Cd were administered subcutaneously (s.c.), orally, or intravenously (i.v.) and subsets of animals were unilaterally castrated or not to evaluate the testes and to collect semen repeatedly from males when adults. There was considerable variability but Cd given at 12 weeks of age in doses of 0.08 mmol/kg s.c., 0.20 mmol/kg orally, and 0.02 mmol/kg i.v. tended to depress sperm output of these males when adults. The 0.02 mmol/kg dose given i.v., 0.4 mmol/kg orally, and 0.16 mmol/kg s.c. were lethal to many animals. Treatment of adults resulted in a generally similar pattern of systemic toxicity, and limited comparisons suggested that testicular sensitivity was slightly less than for young males. Androgenic function usually was maintained, as indicated by normal libido and seminal volumes even in males with reduced spermatogenesis. Necropsies confirmed previous findings of hyperemia, hemorrhaging, necrosis, and destruction of all spermatogenic elements in severely affected males.

Fertility of bull sperm frozen and stored in clarified egg yolk-Tris-glycerol extender.

Egg yolk-Tris-glycerol extender, which is widely used in commercial artificial insemination (AI), was modified by replacing the 20% egg yolk (vol/vol) with a supernatant from egg yolk centrifuged at 50,000 x g for 2 h and then used to cryopreserve bull sperm. Preliminary studies showed that 20% egg yolk interfered with biochemical assays, which could be overcome by centrifugation. In Experiment 1, semen from 4 Holstein bulls was frozen in the experimental Tris extender and compared with the whole milk-glycerol control. A total of 2256 first services resulted in 72.6% 60- to 90-d nonreturns for the control and 71.0% for the Tris extender. In Experiment 2, semen from 10 Holstein bulls was frozen in the experimental Tris extender. Half of the semen was used immediately and half was stored in liquid nitrogen for 1 yr before distribution. The nonreturn rates based on 8878 first services for 7 bulls that completed both parts of the trial were 70.9% initially and 71.6% 1 yr later. This time trend difference of 0.7% was comparable to 0.6% for the AI mean for Holstein sires used at the same time. These fertility results and previous laboratory studies indicate that the conventional egg yolk-Tris-glycerol might be simplified for cryopreserving bull sperm. The experimental Tris extender also was suitable for making biochemical measurements.

Gonadotropin-releasing hormone improves reproductive performance of dairy cows with slow involution of the reproductive tract.

Eighty multiparous Holstein cows were assigned randomly at calving to receive either 100 microg of GnRH or saline 13 or 14 d postpartum (PP). From 4 to 28 d PP the cows' reproductive organs were palpated weekly per rectum, and cows were subclassified within each group as undergoing slow (delayed) cervical and uterine involution (abnormal) or as normal cows. Last milk obtained after removing the milking machine was assayed for progesterone 3 times a week for 120 d PP. Fourteen of the 80 cows were removed from the experiment because of culling or various veterinary treatments of pathologic conditions that could confound analysis of the GnRH treatment effects. As expected, the treatment of normal cows with GnRH had no significant effects on the first estrus or the first estrous cycle PP, on services per conception, days open, or any other reproductive trait measured. However, in the abnormal group of cows receiving saline, first rebreeding after calving was delayed (81 vs. 67 d), fewer were pregnant by 105 d PP (23 vs. 64%), and number of days open was greater (121 vs. 87 d) compared with those receiving GnRH; all were significant (P<.05). Treated abnormal cows were equivalent to the control normal cows. Thus, GnRH given 13 to 14 d PP to cows characterized as undergoing slow involution of the reproductive system, but with no other clinical problems, seems to assist in promoting rapid normal reproductive function. Subsequent losses due to culling were greatly reduced.

Allocation of inner cell mass and trophectoderm cells to the preimplantation blastocyst of the domestic ferret, Mustela putorius furo.

The growth of ferret preimplantation blastocysts in vivo, collected between 156 and 240 hr post coitum, was investigated. A technique, combining immunosurgery and differential fluorochrome staining, was used to discriminate between inner cell mass (ICM) and trophectoderm (TE) cells. Using the stains propidium iodide and bisbenzimide (Hoechst 33342), the ICM was stained blue and the TE was stained pink. The ICM and TE counts for 90 blastocysts, respectively, averaged 25 and 63 at 156 hr and increased exponentially to 2077 and 4137 at 240 hr. The Box-Cox procedure was used for choosing a transformation that minimized the error sum of squares for a linear regression of Y (cell count) on X (time in hr). Logarithmic transformations of the ICM, TE and total cell count gave a good fit, but the following equations obtained by the Box-Cox procedure provided the best fit, where Y is cell count and X is time in hours. For inner cell mass: Y = [(176.06 + 2.45X)/-899.44 + 1]-3.33; trophectoderm: Y = [(301.38 + 14.48X)/-6863.42 + 1]-10; and total: Y = [(2266.97 + 17.0X)/-7837.21 + 1]-5. The R2 values were 0.73, 0.84, and 0.84, respectively. The exponential growth of the ferret embryo during the time interval that measurements were made fits the general pattern described for other mammalian embryos. This report is the first to characterize the pattern of cell allocation and growth in preimplantation blastocysts of the ferret, and the first such report for a carnivore. The pattern of in vivo development provides a standard for judging the quality of in vitro produced and matured ferret embryos and, concomitantly, a means to evaluate culture systems.

Nonsurgical collection and nonsurgical transfer of preimplantation embryos in the domestic rabbit (Oryctolagus cuniculus) and domestic ferret (Mustela putorius furo).

The objective of this study was to develop nonsurgical methods of embryo collection and transfer in domestic rabbits (Oryctolagus cuniculus) and domestic ferrets (Mustela putorius furo) to serve as models for use in mammals in which surgical procedures are the usual means for applying embryo transfer technology. Specially designed transcervical catheters were used together with a fibre optic endoscope to visualize and then catheterize the rabbit and ferret cervices. Five consecutive transcervical uterine flushes in each of eight superovulated female rabbits 78-89 h after an ovulatory injection of LH resulted in the retrieval of 187 embryos, for an average of 23 embryos per rabbit. A total of 116 embryos were nonsurgically transferred to the uteri of ten recipients, and resulted in 23 young (20%). Eight rabbits (80%) produced young with an average litter size of 2.88 (range 1-7). Ten consecutive transcervical uterine flushes in each of 37 female ferrets 145-178 h after an ovulatory injection of hCG resulted in the retrieval of 324 embryos, an average of 8.76 embryos per ferret. A total of 251 embryos from 27 donors were nonsurgically transferred to the uteri of 31 recipients, and resulted in 65 young (26%). Twenty-eight of the recipients (90%) were initially pregnant, as indicated by postpartum necropsies, and twenty-two ferrets (71%) produced young. The average litter size was 2.95 (range 1-7). This is the first report of live births resulting from the nonsurgical collection of embryos from a donor followed by nonsurgical transfer of those same embryos to a synchronous recipient. The methods reported here can serve as models for use in other mammals in which direct visualization and manipulation of the cervix are not possible, and will be particularly useful in endangered species.