R.G. Saacke

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.

Effects of elevated testicular temperature on morphology characteristics of ejaculated spermatozoa in the bovine

Abstract A mild thermal insult to the testes was studied with respect to ejaculated sperm motility and morphology. A 48-h scrotal insulation was administered to 6 young Holstein bulls whose semen was collected by artificial vagina twice in succession at 3-d intervals for 7 wk. For assessment of results, collection days were grouped in the follwing way: Period 1 (control) = Days -6, -3 and 0, where Day 0 = initiation of scrotal insulation after semen collection; Period 2 = Days 3, 6 and 9 (spermatozoa presumed to be in the epididymis or rete testes during scrotal insulation); and Period 3 = Days 12 and 15 through 39 (spermatozoa presumed to be in spermatogenesis during scrotal insulation). Daily sperm output per collection day was 5.3±0.7 × 109 and did not differ across the experimental periods. Moreover, Periods 1 and 2 did not differ in the mean percentage of motility or in the mean percentage of abnormal spermatozoa (69.1±2.5 and 19.6±5.7%, respectively, for Period 1). Morphological change was first noted on Day 12 (47.5±27.4% abnormal) and peaked on Day 18 (86.3±24.3%). Motility depression began on Day 12 and reached a nadir on Day 15 (42.0±9.8%). Bulls varied in the type of abnormal spermatozoa produced and in magnitude of response; however, specific abnormalities appeared in ejaculates in a predictable chronological sequence following scrotal insulation (Day 0). The sequence was: tailless, (Days12 to 15); diadem, (Day 18); pyriform and nuclear vacuoles, (Day 21); knobbed acrosome, (Day 27); and Dag defect, (Day 30).

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.

Relationship of semen quality to sperm transport, fertilization, and embryo quality in ruminants

Abstract Success of a mating is dependent upon both quality and quantity of semen delivered to the female. Males differ not only in fertility at all inseminate dosages, but also in the minimum number of sperm per inseminate required to reach maximum fertility. Thus, two aspects of a males semen quality become important — that which is compensable, where increasing sperm dosage to the female results in a response in fertility, and that which is uncompensable, where differences in fertility among males can no longer be removed by increasing sperm dosage. Compensable traits of semen are those believed to be associated with the inability of sperm to reach the site of fertilization or to initiate the block to polyspermy by penetration of the ovum vestments. Uncompensable traits appear to be associated with the inability of fertilizing sperm to sustain the fertilization process and/or development of the early embryo. It is now important in the evaluation of semen and the male that these two aspects of reproductive deficiency be considered independently. There is also accumulating evidence that low sperm numbers at the site of fertilization (low competition among sperm), due either to problems of male (inseminator) or female origin, may favor fertilization by less competent sperm that may be unable to complete the fertilization process or sustain the young embryo.

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.

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.

Survival of Honey Bee (Hymenoptera: Apidae) Spermatozoa Incubated at Room Temperature from Drones Exposed to Miticides

Abstract We conducted research to examine the potential impacts of coumaphos, fluvalinate, and Apilife VAR (Thymol) on drone honey bee, Apis mellifera L. (Hymenoptera: Apidae), sperm viability over time. Drones were reared in colonies that had been treated with each miticide by using the dose recommended on the label. Drones from each miticide treatment were collected, and semen samples were pooled. The pooled samples from each treatment were subdivided and analyzed for periods of up to 6 wk. Random samples were taken from each treatment (n = 6 pools) over the 6-wk period. Sperm viability was measured using dual-fluorescent staining techniques. The exposure of drones to coumaphos during development and sexual maturation significantly reduced sperm viability for all 6 wk. Sperm viability significantly decreased from the initial sample to week 1 in control colonies, and a significant decrease in sperm viability was observed from week 5 to week 6 in all treatments and control. The potential impacts of these results on queen performance and failure are discussed.

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.

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.

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.