Sperm morphology: Its relevance to compensable and uncompensable traits in semen.

The nature of subfertility due to the male or inseminate is as complex as that of the female. Fertilization failure, and failure in embryogenesis, are both of seminal origin. Males also differ in the number of sperms required to reach their maximum fertilization rate. Males requiring more sperm are considered to have compensable seminal deficiencies. These include a number of known viability and morphology traits (including both abnormal heads and tails) and unknown factors (functional or molecular traits) precluding sperm access to the ovum or ability of the sperm to engage the ovum sufficiently to initiate fertilization and the block to polyspermy. Differences in fertility among males or inseminates independent of sperm dosage are considered uncompensable. These seminal deficiencies are associated with fertilizing sperm that are incompetent to maintain the fertilization process or subsequent embryogenesis (once initiated), with most failures occurring prior to maternal recognition of pregnancy; these sperm would pre-empt fertilization by competent sperm. Evidence now exists supporting the concept that the uncompensable effect is due to chromatin aberrations in morphologically normal or near-normal fertilizing sperm present in abnormal ejaculates (elevated content of abnormal sperm). Thus, sperm morphology may be our best indication for the presence of an uncompensable deficiency, although we have yet to identify the incompetent fertilizing sperm clinically.

Insemination factors related to timed AI in cattle.

Six-day-old bovine ova/embryos were recovered non-surgically and used as biomonitors to evaluate time of artificial insemination. These embryos/ova provided information regarding fertilization status and embryo quality, as well as quantitative and qualitative data regarding associated accessory sperm. Both sperm access to the ovum (addressed by accessory sperm) and fertilization status/embryo quality were important in addressing pregnancy rate for specific intervals from the onset of estrus to insemination. Based on these biomonitors, early insemination failed to achieve optimum pregnancy rate due to inadequate access of sperm to the ovum (i.e., low fertilization rate, manifested by low accessory sperm numbers). However, embryo quality was high in early inseminations, which favors pregnancy. Late insemination failed to achieve optimum pregnancy rate (due to reduced embryo quality), however, sperm access to the ovum was highest. Thus, the selection of an insemination time to achieve optimum pregnancy rate appeared to be a compromise between the two extreme intervals. For timed-AI programs, consideration of the time of ovulation (and its variability) becomes important, in addition to conventional considerations, such as semen handling, site of insemination, and bull selection.

CASIDA AWARD: Thoughts on the components of a graduate education: importance of the professional family.

A major component of a successful graduate education is the student's professional family developed throughout the graduate program. Not only important to the student are those directly guiding him or her, such as the advisor and graduate committee, but also those who directly or indirectly influence the student's competence, philosophy, ethics, outlook, and professional development in the chosen field. A comparison of this professional family with the student's own blood relatives and extended family is discussed with an introduction to the word mishpucha, a Yiddish term describing this large personal family and its influence.

Assessment of pronuclear formation following in vitro fertilization with bovine spermatozoa obtained after thermal insulation of the testis.

This study was conducted to follow the chronology of pronuclear formation in bovine zygotes after in vitro insemination with a population of spermatozoa having abnormal morphology. Semen samples were obtained and cryopreserved from four Holstein bulls before and after a scrotal insulation period of 48 h (Day 0). A pre-insult (Day 5) and a Day 20 post-insult semen sample were evaluated for morphology and used for IVF after standard swim-up sperm separation protocols. Pronuclear formation was scored on subpopulations of presumptive zygotes after they were fixed and stained at 3-h time intervals from 6 to 18 h post in vitro insemination (hpi). Post-thaw morphological evaluation of semen samples revealed a decrease in the percentages of normal spermatozoa in the post-insult samples compared with the pre-insult samples for Bull I (74-22%) and Bull III (68-1%). The sperm penetration rate decreased (P<0.05) between the pre- and post-insult samples for Bulls I (90-76%) and III (92-70%), but was not different for Bulls II (92-90%) and IV (78-85%). The pronuclear formation rates for post-insult zygotes for Bulls II and IV had comparable increases in development over time, whereas there was no increase in the pronuclear development for the zygotes from the post-insult samples for Bulls I and III, and generally a condensed sperm head was observed in the oolemma. At 18 hpi the fertilization rate between the pre- and post-insult samples for Bulls I (51-4%), II (88-75%) and III (94-2%) decreased (P<0.01), but there was no change for Bull IV (66%). In conclusion, we inferred that the failure in normal pronuclear formation was associated with an absence of normal decondensation of the penetrating spermatozoon; this suggested that the effect of morphologically abnormal spermatozoa occurred prior to cleavage, thus limiting early development.

The incidence of apoptosis after IVF with morphologically abnormal bovine spermatozoa.

Normal embryonic development depends on the maintenance of a population of normal healthy cells within each embryo. The aim of this study was to use a combination of apoptotic measures to assess differences in embryo quality after IVF with semen samples with high percentages of abnormal spermatozoa. Semen samples were obtained and cryopreserved from four Holstein bulls before (5 day prior) and after (2 week-post-insult; 2 week-PI and day 20; 3 week-PI) a scrotal insulation period of 48 h (day 0). The swim-up sperm separation method was used. The post-thaw morphology revealed a decrease (P < 0.01) in the percentages of normal spermatozoa in the 3 week-PI samples in comparison with the pre-insult samples for Bulls I and III (74-22.3 and 67.7-0.5%, respectively). The percentage of vacuolated spermatozoa increased significantly for Bull II. After 18 h of sperm-oocyte co-incubation, zygotes were cultured and subpopulations were removed from culture at day 8 and subjected to either the TUNEL or caspase assay. On day 8, caspase intensity increased significantly for both Bull I (217+/-147) and Bull III (229+/-98) for the 3 week-PI embryo groups compared to the equivalent embryo groups for Bull II (98+/-115) and Bull IV (90+/-111). In conclusion, the inability to consistently measure apoptosis with TUNEL alone complicated the assessment of differences in embryo quality. Thus, it is uncertain exactly when during early pre-implantation development the differences in embryo quality are first manifest. Despite discrepancies, our results clearly indicated a difference in the embryo quality between embryos obtained after IVF with semen samples from bulls that had an intense response to scrotal insulation.

Bovine embryo development after IVF with spermatozoa having abnormal morphology.

The study was conducted to evaluate the effects of scrotal insulation on semen samples collected from bulls on embryonic development after IVF. Semen samples were obtained and cryopreserved from four Holstein bulls before and after a scrotal insulation period of 48 h (Day 0). Three types of samples were used for IVF: (1) semen from the test bulls collected 5 d prior to scrotal insulation (pre-insult); (2) semen from Day 13 (2-week post-insult; 2-week PI); and (3) semen from Day 20 (3-week PI). After 18 h of sperm-oocyte co-incubation, the zygotes were cultured for 8 d when a developmental score (0=degenerate, 1=2-cell embryo through 5=blastocyst) was assigned to each embryo. The post-thaw morphological evaluation of sperm samples revealed a decrease (P<0.01) in the percentages of normal spermatozoa in the 3-week PI samples in comparison with the pre-insult samples for Bulls I and III (74-22.3% and 67.7-0.5 %, respectively). The percentage of vacuolated spermatozoa increased significantly for Bull II. The cleavage and blastocyst formation rates and embryo development scores were affected (P<0.01) by the interaction of bull by sample collection time. For Bulls I and III (severe responders) the scrotal insulation effects persisted from the time of cleavage through blastocyst formation. In contrast, the cleavage and blastocyst formation rates for Bulls II and IV were unaffected, despite high percentages of vacuolated spermatozoa present in the post-insult samples for Bull II. In conclusion, the use of scrotal insulation to elevate scrotal temperature was an effective method to obtain semen samples with high percentages of abnormal spermatozoa. The decrease in embryonic development after IVF when using spermatozoa with morphological abnormalities seemed to be multifaceted and related to changes in head morphology.

Effect of time of insemination on number of accessory sperm, fertilization rate, and embryo quality in nonlactating dairy cattle.

Two experiments were conducted to determine the effect of insemination time on number of accessory sperm per embryo (ovum), fertilization rate, and embryo quality. Semen was collected from three fertile Holstein bulls and cryopreserved in egg yolk-citrate-glycerol. In experiment 1, cows were continuously monitored for behavioral estrus by the HeatWatch estrous detection system and were artificially inseminated (AI) with one 0.5-ml straw (25 x 10(6) sperm) at the onset of estrus (AI 0 h), 12 h after onset (AI 12 h), or received natural service at 0 h (Nat 0 h) from one of three bulls. From 150 inseminations, 115 embryos and ova (AI 0 h: n = 39; AI 12 h: n = 39; Nat 0 h: n = 37) were recovered 6 or 7 d after insemination. Fertilization rates differed between treatments (AI 0 h: 67%; AI 12 h: 79%; Nat 0 h: 98%). Median accessory sperm per embryo (ovum) also differed (AI 0 h: 1; AI 12 h: 10; and Nat 0 h: 27) and paralleled the fertilization rate. Embryo quality was not affected by insemination time or natural service. In experiment 2, cows received AI at 0, 12, or 24 h (AI 24 h) after the onset of estrus as determined by HeatWatch. From 154 inseminations, 117 embryos and ova (AI 0 h: n = 39; AI 12 h: n = 39; AI 24 h: n = 39) were recovered 6 or 7 d after insemination. Fertilization rates did not differ in experiment 2 (AI 0 h: 66%; AI 12 h: 74%; AI 24 h: 82%); however, a trend toward a higher fertilization rate accompanied AI 24 h. Median accessory sperm values increased from AI 0 h (1) to AI 24 h (4). Embryo quality declined with AI at increasing intervals after onset of estrus, as percentages of excellent and good, fair and poor, and degenerate embryos were as follows: 77, 15, 8; 52, 38, 10; and 47, 19, 34 for the 0-, 12-, and 24-h inseminations, respectively. Results indicate AI 12 h after the onset of estrus provides a compromise between potential fertilization failure (AI 0 h) and embryo failure (AI 24 h), despite increased accessory sperm per embryo (ovum) after AI 24 h. Artificial insemination 12 h after onset of estrus should optimize fertility of dairy cattle through an acceptable fertilization rate, number of accessory sperm per embryo, and desirable embryo quality.

The effect of time of artificial insemination on fertilization status and embryo quality in superovulated cows.

Thirty nonlactating Holstein cows were superovulated to determine the effect of artificial insemination time on fertilization status and embryo quality. During the luteal phase of the estrous cycle, cows were administered 38 mg FSH-P in a 4-d descending dose regimen. Luteolysis was induced with two injections of prostaglandin on the last day of FSH-P treatment. All cows were continuously monitored for behavioral estrus using the HeatWatch estrus detection system. All cows were inseminated once with one .5-mL straw (50 x 10(6) sperm) at either 0 (n = 10), 12 (n = 10), or 24 h (n = 10) after the first standing event. The elapsed time (mean +/- SD) from the first prostaglandin dose to the first standing event was 39.4 h +/- 7.7 h. The (mean +/- SD) duration of behavioral estrus was 13.2 h +/- 4.1 h. The (mean +/- SD) number of standing events was 27 +/- 17. Five hundred twenty-nine embryos and ova were recovered nonsurgically 6 d after insemination. Fertilization rates were 29 (0 h), 60 (12 h), and 81% (24 h) (P < .01). Percentages of excellent and good, fair and poor, and degenerate embryos were not different (P > .05). Percentages of embryos with accessory sperm were 5 (0 h), 8 (12 h), and 41 (24 h) and differed between the 0 and 24 h and the 12 and 24 h inseminations (P < .01). Artificial insemination of superovulated, nonlactating Holstein cattle 24 h after onset of estrus increased fertilization rate and percentage of embryos with accessory sperm compared with insemination at 0 or 12 h after onset of estrus. Embryo quality was not affected by time of insemination.

Relationship of seminal traits and insemination time to fertilization rate and embryo quality.

The nature of subfertility due to the male or inseminate is as complex as that of the female. Fertilization failure or failure in embryogenesis are both documented to be of seminal origin. Males also differ in the numbers of sperm required to reach their maximum fertilization rate. Males requiring more sperm would be considered to have compensable seminal deficiencies. These include a number of known (viability and morphology) and unknown factors (functional or molecular traits) precluding sperm access to the ovum or ability to engage the ovum sufficiently to initiate fertilization and the block to polyspermy. Differences in fertility among males or inseminates independent of sperm dosage are considered uncompensable. These deficiencies would be associated with fertilizing sperm that are incompetent to maintain the fertilization process or subsequent embryogenesis once initiated, with most failures occurring prior to maternal recognition of pregnancy. Such sperm would preempt fertilization by competent sperm. Chromatin aberrations in morphologically normal or near normal spermatozoa from abnormal semen samples appear to be the best candidates for the uncompensable deficiency. However, recognition of uncompensable or incompetent fertilizing sperm has not been achieved. Six-day-old non-surgically recovered bovine ova/embryos have been used to evaluate compensable and uncompensable seminal deficiencies as well as to test reproductive strategies. These ova/embryos provide information on fertilization status and embryo quality as well as quantitative and qualitative data regarding associated accessory sperm. Thus, they permit the separation of reproductive failure by fertilization from that by embryonic development. Accessory sperm number is positively associated with both fertilization rate and embryonic quality. Early insemination results in low fertilization rates (low accessory sperm number), but good embryo quality, whereas, late insemination results in high fertilization rates (high accessory sperm number), but poor embryo quality. Additional studies will be necessary to substantiate this model; however, if true, future research designed to improve results to artificial insemination should be tested by breeding early in estrus where sperm viability is most limiting and embryo quality is best.

Effect of long-term immunization against inhibin on sperm output in bulls.

To determine the effect of neutralization of inhibin on sperm output, 12 Holstein bulls were paired by birth date and weight on Day 1 of age. Each bull was actively immunized against bovine inhibin alpha1-26 gly-tyr (bINH) conjugated to human alpha globulin (HAG, n = 6 bulls) or HAG alone (controls, n = 6) at 60 days of age; booster immunizations were administered at 90, 104, 124, 270, and 395 days of age. Body weights and scrotal circumferences were measured at the time of primary immunization and at 10 days after each booster. In addition, jugular blood was obtained at 60, 70, 100, 114, 134, 280, and 405 days of age, during the 3-wk sperm collection period, and during a 6-h blood-sampling period after sperm collection to determine bINH antibody titer and concentrations of FSH, LH, testosterone, and estradiol. Beginning at 405 days of age, sperm output was measured 3 days/wk for 3 wk with two successive ejaculates collected each day for a total of 18 ejaculates per bull. During Days 60-405 of age, the increase in titer of bINH antibodies, scrotal circumference, and serum concentration of FSH was greater (p < 0.01) for the bINH-immunized compared with control bulls. There were significant (p < 0.01) pair x treatment interactions for sperm output and serum FSH and LH concentrations. Specifically, bINH-immunized bulls for four of the six pairs had nearly 50% greater serum FSH concentrations and sperm output. For the remaining two pairs, sperm output was lower and FSH was either lower or only marginally higher in the bINH-immunized bulls compared with controls. Also, the control bulls for the two remaining pairs produced more sperm than all but one bINH-immunized bull, and had markedly higher serum LH concentrations than all other bulls. To summarize, enhancement of sperm output after immunization against inhibin depends on the subsequent increment in FSH concentrations. We conclude that inhibin suppresses spermatogenesis. Thus, methods to immunoneutralize inhibin may have merit as a therapeutic route to enhance sperm production in reproductively maturing bulls.

Effect of a deep uterine insemination on spermatozoal accessibility to the ovum in cattle: a competitive insemination study.

A competitive insemination study was conducted to determine the effect of a deep uterine insemination on accessory sperm number per embryo in cattle. Cryopreserved semen of a fertile bull characterized by spermatozoa with a semi-flattened region of the anterior sperm head (marked bull) was matched with cryopreserved semen from an unmarked bull having spermatozoa with a conventional head shape. Using 0.25-mL French straws and a side delivery embryo transfer device, deep uterine insemination (0.125 mL deposited in each horn) was performed 2 cm from the uterotubal junction. Immediately after, the uterine body was artificially inseminated using semen (0.25 mL) from an alternate bull and a conventional insemination device. The complete dose (both inseminations) was 50x10(6) total sperm cells consisting of an equal number of spermatozoa from each bull. Single ovulating cows (n = 95) were inseminated at random with either the unmarked semen in the uterine body and marked semen in the uterine horn, or the unmarked semen in the uterine horn and marked semen in the uterine body. Sixty-one embryos(ova) were recovered nonsurgically 6 d post insemination, of which 40 were fertilized and contained accessory spermatozoa. The ratio and total number of accessory spermatozoa recovered was different among treatments: 62:38 (326) for the unmarked semen in the uterine body and marked semen in the uterine horn, and 72:28 (454) for the unmarked semen in the uterine horn and marked semen in the uterine body (P<0.05). Deep uterine insemination using this semen in a split dose and a side delivery device favors accessibility of spermatozoa to the ovum compared with conventional uterine body insemination.

Can spermatozoa with abnormal heads gain access to the ovum in artificially inseminated super- and single-ovulating cattle?

The collective efficiency of barriers in the female tract against spermatozoa with abnormal heads was studied. In Experiment 1, Day 6 ova/embryos were recovered nonsurgically from superovulated (n = 24) and single-ovulating (n = 44) cows following artificial insemination with semen of bulls selected for normal spermatozoal motility (> or = 50%) and high content (> 30%) of spermatozoa with misshapen heads, random nuclear vacuoles or the diadem defect. To assess characteristics of spermatozoa capable of traversing barriers in the female tract, accessory spermatozoa were classified morphologically (x 1250) and compared with those of the inseminate. Superovulated cows proved inadequate for assessment of accessory spermatozoa due to evidence of poor sperm retention in the zona pellucida; thus, only single-ovulating cows were used. Accessory spermatozoa (n = 479) from 31 ova/embryos recovered from 44 cows were more normal in head shape than those in the inseminate (76 vs 62%; P < 0.05). Spermatozoa with normal head shape, but with nuclear vacuoles appeared as accessory spermatozoa at the same frequency as they were found in the inseminate (20 vs 17%, respectively). Only sperm cells with subtly misshapen heads appeared as accessory spermatozoa. In Experiment 2, semen pooled from 4 bulls having large numbers of spermatozoa exhibiting a gradation from severely asymmetrically misshapen heads to subtly misshapen heads was evaluated. Again, the accessory sperm population (960 sperm cells recovered from 64 ova/embryos) was enriched with spermatozoa of normal head shape relative to the inseminate (53 vs 26%, respectively; P < 0.05). Sperm cells with only nuclear vacuoles and those with subtly misshapen heads were not different between the accessory and inseminate populations (11 vs 8%, and 20 vs 25%, respectively). We conclude that morphologically abnormal spermatozoa are excluded from the accessory sperm population based upon severity of head shape distortion.

Chromatin structural changes in sperm after scrotal insulation of Holstein bulls.

The reported effects on semen quality ascribed to testicular heat stress generally relate to traits impacting sperm transport and fertilizing ability but not to the genetic material contained by the sperm. To characterize the effects of testicular heat stress on sperm chromatin, susceptibility of DNA in sperm nuclear chromatin to in situ acid denaturation was measured by flow cytometry after staining with acridine orange using the sperm chromatin structure assay (SCSA). Semen was collected from Holstein bulls at 3-day intervals, before and after 48-hour scrotal insulation, until the morphologically abnormal sperm content in raw semen exceeded 50%. After cryopreservation in egg yolk-citrate extender, semen was thawed and sampled during incubation in vitro at 38.5 degrees C. Overall, SCSA results showed that chromatin susceptibility to denaturation was increased for sperm collected post- vs. preinsulation and was more pronounced for sperm presumably in the testes during insulation than for those sperm presumably in the epididymides. Increased susceptibility was detected as early as the first collection postinsulation; however, chromatin of sperm presumably in the proximal epididymis during insulation did not appear to have been detrimentally affected. Chromatin susceptibility to denaturation increased with increased incubation time in vitro, but the rate of change in susceptibility during incubation did not differ among pre- vs. postinsulation specimens. We conclude that elevated scrotal temperatures adversely affect both epididymal and testicular sperm by reducing sperm chromatin stability. The effects of heat stress on the chromatin of epididymal sperm were more subtle than those exhibited by testicular sperm but detectable within close proximity to the heat stress event.

Ejaculation increases scrotal surface temperature in bulls with intact epididymides.

The objective of the study was to determine the effects of ejaculation on scrotal surface temperature (SST) measured with infrared thermography in bulls. In 18 Holstein bulls (18 mo old), sexual stimulation and spontaneous ejaculation (into an artificial vagina) increased SST at the bottom of the scrotum (0.9 degrees C; P < 0.0001). In 11 Angus bulls (1 yr old) electroejaculation increased both bottom and average SST (1.7 degrees C; P < 0.005 and 0.9 degrees C, P < 0.05), while in 12 Simmental cross bulls (2 yr old) electroejaculation significantly increased top, bottom and average SST (1.0, 1.2 and 1.1 degrees C, respectively). However, there was no significant increase in SST following electroejaculation in 15 Simmental cross bulls (2 yr old) with caudal epididectomies. The increase in SST was attributed to a localized increase in SST over the cauda epididymides, perhaps due to heat produced by contraction of the cauda epididymides during ejaculation. The results support the hypothesis that spontaneous ejaculation or electroejaculation increases SST and that this response is mediated by the cauda epididymides. Infrared thermography of the scrotum for evaluation of scrotal/testicular thermorégulation for clinical or research purposes should be performed before semen collection since thermography conducted soon after ejaculation may be misleading.

Insulating the scrotal neck affects semen quality and scrotal/testicular temperatures in the bull.

Nine Simmental X Angus bulls (2-yr of age) were used in 2 experiments. In Experiment 1, the scrotal neck was insulated (from Day 1 to Day 8) in 5 bulls, and semen was collected from all 9 bulls by electroejaculation approximately every 3 d until Day 35. Bulls with insulated scrotal necks had lower percentages of normal spermatozoa (P < 0.08) and higher percentages of spermatozoa with head defects (P < 0.06) or droplets (P < 0.08) than the untreated bulls. There was a time-by-treatment interaction (P < 0.04) for midpiece defects; the incidence was higher (P < 0.05) in the insulated than noninsulated bulls from Day 5 to Day 32. Spermatozoa within the epididymis or at the acrosome phase during insulation appeared to be the most affected. Compared with the noninsulated bulls, the insulated bulls had twice as many (P < 0.02) spermatozoa with midpiece defects and 4 times as many (not significant) with droplets on Day 5, fewer (P < 0.04) normal spermatozoa and 3 times as many with midpiece defects (P < 0.05) and with droplets (not significant) on Day 8, fewer (P < 0.02) normal spermatozoa on Days 15 and 18, and more sperm cells (P < 0.05) with head defects on Days 18 and 21. In Experiment 2, scrotal subcutaneous temperature (SQT; degrees C, mean +/- SE) prior to and after the scrotal neck had been insulated for 48 h in all 9 bulls was 30.4 +/- 0.7 and 32.4 +/- 0.6 (P < 0.01) at the top, 30.3 +/- 0.7 and 31.8 +/- 0.6 (P < 0.03) at the middle, and 30.2 +/- 0.8 and 30.7 +/- 0.6 (P < 0.05) at the bottom of the scrotum. Concurrently, there was an increase (0.9 degrees C) in intratesticular temperature (ITT) at the top (P < 0.07), middle (P < 0.04), and bottom (P < 0.04) of the testes. Scrotal surface temperature (SST) prior to and after the scrotal neck had been insulated for 24 h was 29.2 +/- 0.7 and 28.2 +/- 0.4 (P < 0.05) at the top of the scrotum and 24.7 +/- 0.6 and 25.3 +/- 0.7 (not significant) at the bottom, resulting in SST gradients of 4.6 +/- 0.6 and 2.9 +/- 0.5, respectively (P < 0.05). However, after the scrotal neck had been insulated for 48 h, none of the SST end points were significantly different from those prior to insulation. It appears that compensatory thermoregulatory mechanisms restored SST but were not able to restore SQT and ITT. Insulation of the scrotal neck affected SST, SQT, ITT and semen quality, emphasizing the importance of the scrotal neck in scrotal/testicular thermoregulation.

Variation among inbred and linecross mice in response to fescue toxicosis.

Variation in response to fescue toxicosis was examined in inbred and linecross mice. In Exp. 1, exposure to a 50% endophyte-infected tall fescue diet (E+) reduced ADG of males from six inbred lines, but ADG of males from one line was modestly higher on E+. Lines differed (P < .01) for reproductive organ weight, but the diet x line interaction was not significant. In Exp. 2, an apparently susceptible (C57) and an apparently resistant line (FVB) were mated to produce inbred and linecross offspring. The reduction in weight gain caused by the E+ diet did not differ significantly among the genetic groups. In Exp. 3, C57 and C57 backcrosses had smaller reductions in ADG during E+ vs control feeding periods than FVB and FVB backcrosses (P < .10). In Exp. 4, the E+ diet reduced litter size of mates of C57 males by one pup, whereas litter size of mates of FVB males was four pups larger (interaction P = .07). Neither diet, line, nor their interaction affected male reproductive organ weights or tissue proportions in testis cross-sections. In Exp. 5, the E+ diet did not affect weight gain of C57 or FVB males, but effects of the E+ diet on litter size of mates were similar to those in Exp. 4. Percentage of abnormal sperm was increased in C57 males on the E+ diet but decreased in FVB males (Exp. 5). Differences among inbred lines in susceptibility to fescue toxicosis may depend on severity of the challenge and life cycle stage when the challenge is imposed.

Technology and applications for encapsulated spermatozoa.

A technique for microencapsulation of bovine spermatozoa has been developed with minimal spermatozoal injury and thus, a potential use in artificial insemination (AI). Membranes made of the following polymers have proven best: poly-l-lysine, polyarginine, polyvinylamine, and protamine sulfate. Successful encapsulation has been achieved for capsules ranging in size from 0.75 to 1.5 mm, and sperm concentrations from 45 to 180 x 10(6) cells/ml. Successful buffers include Cornell University Extender and egg yolk citrate - glycerol (maximum 10% v/v egg yolk for normal capsular shape). Capsule fragility (ability to rupture under aging and physical stress) was negatively related to membrane thickness which ranges from 1.92 microm to 5.32 microm (controlled by polymer concentration, molecular weight, and exposure time) and positively to concentration of sperm encapsulated. On delivery to the porcine reproductive tract, the capsule constructed of poly-l-lysine membranes ruptured between 12 and 24 hr after insemination. Heterospermic studies have shown that encapsulated sperm are capable of fertilization in vivo, but are at a disadvantage to unencapsulated sperm when AI is at conventional times following detection of estrus.

Microencapsulation of bovine spermatozoa for use in artificial insemination: a review.

A technique for microencapsulation of bovine spermatozoa has been developed with minimal spermatozoal injury and thus of potential use in artificial insemination. The polymers poly-l-lysine, polyvinylamine and protamine sulfate have proven best for membranes. Encapsulation has been successful with capsules ranging in size from 0.75 to 1.5 mm, and with sperm concentrations from 45 to 180 x 10(6) cells mL-1. Successful extenders include CUE, CAPROGEN, and egg yolk-citrate-glycerol (maximum 10% v/v egg yolk for normal capsular shape). Capsule fragility (ability to rupture under ageing and physical stress) is negatively related to membrane thickness which ranges from 1.92 to 5.32 microns (depending on the concentration of polymer used) and positively related to concentration of sperm encapsulated. Heterospermic studies have shown that encapsulated sperm are capable of fertilization in vivo, but are at a disadvantage to unencapsulated sperm when cows are inseminated at conventional times. Uterine retention of inseminates is favoured by capsules having a 'sticky' membrane. Using current procedures, preliminary homospermic fertility studies indicate that sperm encapsulated with poly-l-lysine or protamine sulfate may achieve normal fertility.

Effect of freezing semen and dosage of sperm on number of accessory sperm, fertility, and embryo quality in artificially inseminated cattle.

This experiment was conducted to determine whether use of fresh or frozen semen at either 20 x 10(6) (low) or 100 x 10(6) (high) sperm per dose affects the number of accessory sperm and fertilization status/embryo quality as determined from ova/embryos recovered nonsurgically 6 d after insemination. Ejaculates of four bulls were split and prepared for use as fresh or frozen semen at either the high or low dose. From 129 inseminations to normally cycling cows, 98 ova/embryos were recovered. To reduce male effects, ova/embryos used were randomly balanced across treatments, by ejaculate within bull for evaluation of frozen vs fresh semen (n = 80) and by bull for evaluation of high vs low dosage treatments (n = 76). Distribution of accessory sperm was highly skewed downward; thus, median values were more meaningful than means. Freezing semen had no significant effect on fertility status/embryo quality or number of accessory sperm at either dosage. Increasing dosage improved the number of accessory sperm per ovum or embryo (median value) and fertility status/embryo quality (P < .05). Mean +/- SD and median values for accessory sperm were 37.8 +/- 38.3 and 27.5; 28.9 +/- 62.8 and 3.0 for the high and low dose, respectively. Percentage of unfertilized ova, degenerate embryos, and embryos classified poor to fair and good to excellent were 3, 5, 24, 68, and 21, 16, 18, 45, for the high and low dose, respectively. We conclude that number of accessory sperm and fertility status/embryo quality respond favorably to increased dosage of semen and that freezing semen in this study was not detrimental to these parameters.

Accessory sperm numbers for cattle inseminated with protamine sulfate microcapsules.

A study utilizing two morphologically distinct sperm populations in a heterospermic trial was conducted to evaluate the effect of microencapsulation on accessory sperm number in artificially inseminated cattle. Inseminates consisted of an equal number (100 x 10(6) of morphologically normal and tapered sperm. In reciprocal inseminates, one of the two sperm types was encapsulated in protamine sulfate microcapsules, and the other type was unencapsulated. Insemination of both sperm types that were unencapsulated served as the control. Thirty-one embryos were nonsurgically recovered 6 d postinsemination, and accessory sperm within the zona pellucida were counted and morphologically classified as normal or tapered. Microencapsulated sperm contributed 25.7% of the accessory sperm obtained for inseminates containing microcapsules. Microencapsulation did not affect the total number of accessory sperm per embryo; however, encapsulation did reduce accessory sperm for the specific sperm type encapsulated. The proportion of morphologically normal to total accessory sperm recovered were 65.8% when both sperm types were unencapsulated, 35.4% when normal sperm were encapsulated, and 80.6% when the tapered sperm were encapsulated. High accessory sperm numbers per embryo were related to embryo quality (P less than .01). Mean accessory sperm and standard deviations for degenerate embryos and for embryos classified fair to poor, good, and excellent were 1.7 +/- 2.9, 11.1 +/- 8.6 24.9 +/- 33.9, and 84.3 +/- 58.3. We conclude that sperm inseminated in protamine sulfate microcapsules can be released and can attach to the zona pelucida of ova in vivo. Microencapsulation failed to increase accessory sperm numbers under conditions of this experiment in which insemination was 12 h after estrus was first detected.