Bull sperm surface "craters" and other aspects of semen quality

Semen from 200 Holstein bulls in an artificial insemination center was examined for the frequency of craters on the surface of sperm heads, as visualized with the aid of differential interference contrast microscopy. Semen from 100 of these bulls was examined in more detail in 2 experiments by staining with eosin-aniline blue to determine the relationship of unstained spermatozoa, and spermatozoa with normal acrosomes with apical ridges to the incidence of craters and fertility. Only 3 of 100 bulls had a substantial incidence of craters (15 to 23%), whereas the average of the other 97 bulls in 2 experiments was 1 to 3%. The percentage of sperm cells with craters was correlated (P < 0.05) with the percentage of unstained spermatozoa (r = -0.29 and sperm cells with normal acrosomes (r = -0.52) but was not significantly correlated (r = -0.24) with the nonreturn rate. One bull with many sperm cells with craters was slaughtered, and the epididymal spermatozoa were examined. The high incidence of sperm cells with craters was limited to one side, with the testis on that side having 2 Sertoli cell tumors. The remaining 2 bulls as well as one other that produced 16% of sperm cells with craters did so only temporarily. Within a few months crater sperm production had decreased and semen quality increased. The condition usually appears to be transitory, presumably due to temporary stress.     

Sperm chromatin structure assay of bulls qualified for artificial insemination

The goal of our study was to find the relationship between fertility of bulls qualified for AI and the percentage of spermatozoa with abnormal chromatin structure as an independent parameter. We used the frozen semen of 8 mature bulls from one AI center. Each bull was represented by 3 ejaculates collected with at least 2-week intervals. Bull fertility was calculated on the basis of non-return ratio and was expressed as a scale where 100 points represented the average fertility of all the AI center's bulls. Bulls with lower or higher fertility received a lower or higher score respectively. Fertility scores of bulls used in the study ranged from 83 to 104 . Semen was processed according to the SCSA (sperm chromatin structure assay) method and was analyzed by flow cytometry. "Artificial" alpha(t) (alpha(t)=red/green+red fluorescence) and red fluorescence histograms were used for calculation of COMPalpha(t), SDalpha(t), %Red, %PeakR and MeanR parameters. The percentage of spermatozoa with abnormal chromatin ranged from 1.2% to 23.8%. A large variation among ejaculates was found for bulls with lower fertility. Fertility correlated significantly with COMPalpha(t) (-0.50, P < 0.05), SDalpha(t) (-0.55, P < 0.01), %Red (-0.53, P < 0.01), %PeakR (-0.58, P < 0.01) and MeanR (-0.45, P < 0.05). The SCSA method has a practical application in analyzing spermatogenesis disorders in bulls. If regularly applied, it allows us to identify and eliminate ejaculates with a high level of sperm chromatin abnormalities.     

Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility.

The present study was conducted to detect sperm apoptosis in fresh and frozen semen and to determine its relationship with bull fertility. Three ejaculates were collected from five breeding bulls with different fertility levels and were cryopreserved using standard methods. Two flow cytometric methods were employed to measure apoptosis: an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labeled Annexin V and propidium iodide (PI), and an assay for nicked DNA using bromodeoxyuridine (BrdU), terminal deoxynucleotidyl transferase, and fluorescein-labeled anti-BrdU monoclonal antibody. Both assays showed that fresh sperm contained 10%-20% apoptotic sperm. Significant differences in the percentage of apoptotic sperm were observed among the bulls. Cryopreservation induced translocation of PS to the outer leaflet of the plasma membrane and caused most of the necrotic cells in fresh sperm to disintegrate. Bull fertility was significantly related to the percentage of necrotic or viable sperm in fresh semen as detected by the Annexin V/PI assay, to the number of apoptotic sperm in fresh semen as detected by the TUNEL assay, and to the level of chromatin or DNA condensation as detected by PI staining. The present study suggests that the presence of apoptotic spermatozoa in fresh semen could be one of the reasons for poor fertility in breeding bulls.     

Sperm accumulation in the ampullae and cauda epididymides of bulls

Bulls that appear to have an abnormality of sperm transport accumulate large numbers of senescent sperm in the excurrent ducts of the reproductive tract. Six bulls that accumulated sperm were used to determine the number of physiological ejaculations required to deplete accumulated senescent sperm, semen traits during depletion, the period of time to re-accumulate senescent sperm after depletion, the sites of sperm accumulation, and the effect of sperm accumulation on fertility during natural service. Semen was collected from three bulls (HH, PH1, and CH1) three times daily using internal artificial vaginas placed in cows in estrus. These bulls started to produce semen with >or=70% morphologically normal sperm on the third, fifth, and seventh day, respectively. The percentage of live sperm increased from 5% to 68%, 5% to 63%, and 18% to 68% in HH, PH1, and CH1, respectively. Two weeks later the same bulls were electroejaculated every second day for five electroejaculations to deplete stores of senescent sperm. Each time, electroejaculation was continued until the semen produced had a dilute appearance. The three bulls re-accumulated senescent sperm after 1 month of sexual rest. After re-accumulation of senescent sperm, the total volumes of semen in both ampullae recovered at slaughter from HH, PH1, and CH1 were 5.0, 5.0, and 9.5 ml, respectively. The volumes of semen in ampullae recovered at slaughter from two control bulls (RA and CHC) were 1.7 and 1.9 ml, respectively. The number of sperm recovered from both cauda epididymides of HH, PH1, CH1, RA and CHC was 37.3x10(9), 23.3x10(9), 15.0x10(9), 6.9x10(9), and 7.4x10(9), respectively. Bull CH1 and a fourth bull (LM) that also accumulated sperm, started to produce semen with >or=70% morphologically normal sperm, and >or=60% progressively motile sperm on the third electroejaculation after depletion of senescent sperm by repeated electroejaculations. Pregnancy rates achieved by two bulls that accumulated senescent sperm (CH2 and PH2) were less (P<0.05) during the first week of a 21-day breeding period and there was a tendency for lesser pregnancy rates at the end of the breeding period when compared with two normal control bulls. The present study indicates that bulls that accumulate senescent sperm may achieve greater pregnancy rates approximately 1 week after beginning a period of frequent ejaculation. Re-accumulation of an increased percentage of senescent sperm would likely occur after 1 month of sexual rest.     

Fertility of cryopreserved sperm in three bulls with different Robertsonian translocations

The fertility of three bulls carrying different Robertsonian translocations (rob(1;29), rob(14;17) and rob(26;29)) was evaluated. Oocytes-cumulus complexes obtained from slaughterhouse-derived ovaries were matured and then fertilised in vitro with frozen/thawed seminal material from the above mentioned subjects, and from control bulls with normal karyotype. An assessment was first made of the concentration, vitality and acrosome integrity of the seminal material to be sure that possible differences in the results of the in vitro fertilisation experiments were not due to seminal material quality. The results of the experiments, evaluated by the percentage of cleaved embryos and blastocysts per cleaved embryo, indicated that the three bulls carrying Robertsonian translocations had similar fertilising power and semen qualitative parameters to the controls. These data suggest that neither gametogenesys impairment nor decreased spermatozoa fertilising capacity is responsible for the reduced fertility in bulls with Robertsonian translocations. What the data do confirm is that the observed in vivo hypofertility for karyologically abnormal bulls is mainly due to early embryonic mortality.     

A new reciprocal translocation in a subfertile bull

Three bulls of the Montbéliarde breed that exhibited fertility rates lower than 30% following more than 400 artificial inseminations were examined. Semen quality (sperm motility and morphology) from these bulls was normal. Fertilizing ability estimated from in vitro embryo production results was studied for two of them. In vitro production rate was very low for one bull (A) and normal for the other (B). Cytogenetic analyses were carried out on the three bulls using chromosome banding techniques. These analyses revealed a reciprocal translocation (12;17)(q22;q14) in bull B. Based on family analyses, the hypothesis of a de novo origin of this rearrangement is proposed.     

牛分离精子对其IVF胚胎染色体影响的研究

本研究采用传统的细胞遗传学方法,研究了由流式细胞仪分离的、染色未分离的及作为对照用的未染色未分离的分别来自于3头公牛的精子IVF（in vitro fertilization, IVF)后产生的6~8 d囊胚的染色体异常情况,以确定流式细胞仪分离精子的过程及染色对胚胎染色体异常的影响。结果显示,分离精子、染色未分离精子和未染色未分离精子的胚胎中,染色体组成为异常,即嵌合体的胚胎分别占40.7%（59/145）、35.8%（38/106）和37.0%（37/100）,三者染色体异常的比例无显著差异。胚胎染色体异常的频率在不同公牛之间存在差异（33.0% 比 44.6%）（p<0.05）。本研究的结果证明,染料和分离过程没有影响精子的DNA进而影响胚胎的染色体组成;胚胎染色体异常的频率在不同公牛之间存在差异。     

Use of semen from a bull heterozygous for the 1 29 translocation in an IVF program

In cattle, a translocation of the Robertsonian type between the largest and smallest chromosome leads to a reduction in fertility. This is substantiated by reduced nonreturn rates in daughter groups of bulls carrying the 1 29 translocation and in the heterozygous bulls themselves. This reduction in fertility is thought to be due to the early death of embryos with unbalanced karyotypes. The influence of semen from a bull known to be heterozygous for the 1 29 translocation on the outcome of a bovine IVF program was investigated. There was a significant difference (P<0.005) in terms of cleavage rate (59.8 vs 71.1%) and blastocyst rate (12.0 vs 20.0%) between the carrier and control bull, respectively. There was no difference in blastocyst quality as measured by cell number. The results observed in vitro are consistent with the field fertility records of the 2 bulls in terms of nonreturn rates (59.2 vs 70.6%, for the carrier and control bull, respectively).     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: I. A fertility assay for fresh semen

Fresh sperm from five bulls having nonreturn rates ranging from 48% to 77% were treated with 15.7, 21.0, 26.2, 31.5, 36.7, and 42.0 μM dilauroylphosphatidylcholine (PC12) to induce the sperm acrosome reaction (AR). Treated sperm were incubated 3 hr with zona-free hamster eggs at 39°C prior to fixation. The eggs were then stained and examined for sperm penetration. Differences in the percentages of motile sperm and of sperm exhibiting an AR among bulls were small when compared on a within-liposome-concentration basis. Increasing the PC12 concentration from 15.7 μM to 42.0 μM increased the percentage of sperm exhibiting an AR for all bulls. At the lowest lipid concentration (15.7 μM), the percentage of eggs penetrated by sperm from the five bulls was 6% to 36%, with 0% in controls. When sperm were incubated with increasing lipid concentrations, the egg penetration rate increased to over 80%, and the total number of sperm increased to over 100 per 36 eggs in each treatment for every bull. These penetration rates decreased at the highest lipid concentration. A correlation between the PC12 concentration maximizing egg penetration and the nonreturn rate of ?.63 was found. The correlation between the PC12 concentration maximizing the total number of penetrated sperm per treatment and the bull nonreturn rate was ?.96. It was concluded that PC 12 liposomes induce the AR in bull spermatozoa, which enables them to penetrate zona-free hamster eggs. High fertility bulls required less lipid to induce the AR than did lower fertility bulls. Consequently, this assay of fresh semen could provide a laboratory method to estimate the fertility of a bull.     

X-linked mental retardation with the fragile X. A study of 15 families

Summary The coinical and cytogenetic features of 15 families with mental retardation linked to the fragile site on the X chromosome are presented.The 15 propositi were all prepubertal, and one was a girl. Although the clinical picture varied in severity, it was sufficiently constant to suggest the diagnosis from the facial features and the encephalopathy with language retardation and disturbed behavior. Macroorchidism was not seen before puberty.The fragile X chromosome was found in seven of the nine mothers studied and in two mildly retarded sisters and has also been demonstrated in fibroblasts in eleven subjects with the abnormality.Supported by grants from INSERM (ATP 79-110)     

Efforts to correlate laboratory with field observations on bull sperm fertility

This work represents efforts towards development of the zona-free hamster ovum sperm penetration assay for predicting relative levels of fertility for semen from individual bulls. Results reported here followed insemination of hamster vitelli with bull sperm, after frozen storage, with observations of sperm acrosomes and parallel inseminations of more than 1000 cows with semen from each bull. The average 75-day non-return rate for the four bulls was 74.0% (range 71.6 to 75.6). Laboratory studies indicated the following: the percentage of sperm with intact acrosomes varied from 55 to 73 between bulls, the percentage of motile sperm varied from 41 to 64, the percentage of sperm with progressive motility ranged from 24 to 40, the number of sperm interacting per (zona-free hamster) ovum ranged from 1.6 to 3.8, the number of sperm attached per ovum ranged from 1.4 to 2.9, the number of sperm within each penetrated ovum ranged from 1.5 to 1.8, the percentage of ova interacting with sperm ranged from 76 to 92, the percentage of ova penetrated ranged from 62 to 85, and the percentage of ova with male pronuclei ranged from 33 to 49. Although predictive ranking in the laboratory of these bulls with less than 4% variation in fertility levels was not possible, the zone-free hamster ovum test could be useful in identifying potentially subfertile bulls before they enter a young sire-sampling program.     

The relationship between breeding soundness evaluation and fertility of beef bulls under group mating at pasture

The relationships between natural service fertility of beef bulls and the components of breeding soundness evaluation, age, preweaning average daily gain, yearling weight and scores of two libido tests were studied in two seedstock herds. In one of the herds (YBH), 15 yearling bulls were rotated during six-week breeding season so that each group of five bulls served the cows for a period of one week and rested for two weeks before the next exposure. Five older bulls (one 3-year old and four 2-year old) were assigned to the other herd (MBH) during the entire breeding season (six weeks). The progeny of each bull were identified by blood typing.Calf crop was 6.4% higher (P=0.14) in the MBH compared with that in the YBH. There were large differences in fertility between the bulls in the MBH. The 3-year old bull sired 40.9% of the calves, presumably due to his high social dominance order. The third group of yearling bulls, which served the herd during the third and sixth weeks of breeding, sired the highest number of calves (49.4%) compared with the other two groups (28.4% and 22.2%). In each of the groups one and two of the yearling bulls, one bull did not produce any progeny, while another one sired 44% of the calves. The correlation coefficients between fertility of the bulls and the traits used to predict their fertilizing capacity were generally small and inconsistent in different groups, when the effect of age in the mixed age group was removed. The correlation coefficients of bull fertility with scrotal circumference and reproductive system score were higher and more consistent in different groups as compared with the other traits studied.     

Assessment of the fertilizing potential of frozen bovine spermatozoa by in vitro induction of acrosome reactions with calcium ionophore (A23187)

The relationship between nonreturn rates of bulls in a commercial artificial insemination program and in vitro induction of acrosome reactions in frozen-thawed spermatozoa by the calcium ionophore, A23187, was investigated. Washed spermatozoa from 3 to 5 ejaculates, collected from each of 23 Holstein bulls, were incubated for 1 h with 1 microM A23187. Acrosome reactions were determined by fluorescence microscopy. The percentage of increase in acrosome reaction in the ionophore-treated compared with control samples was significantly correlated to the 90-d nonreturn rate of the bulls (r = 0.86; P < 0.001). In a second experiment, a significant correlation was obtained between the fertility of bulls predicted on the basis of induced acrosome reaction and achieved 90-d nonreturn rate (r = 0.84; P < 0.005). No other assessments of semen quality (post-freezing motility, percentage of morphologically normal spermatozoa) was significantly correlated with fertility. Finally, the regression between acrosome reaction induction obtained from young bulls was used to predict the fertility of mature bulls whose semen was in widespread use (actual versus predicted nonreturn rate, r = 0.88; P < 0.0001).     

Embryo quality and andrological study of two subfertile bulls versus five control bulls with normal fertility

Andrological studies and embryo morphology evaluation of superovulated cows were performed on 2 randomly selected subfertile dairy bulls whose semen was used for artificial insemination and on 5 control bulls with normal fertility. Neither sperm motility studies, nor sperm morphology or testicular measurements differed between the subfertile and the control bulls. Altogether 315 ova were recovered from 41 superovulated cows inseminated with semen collected from either the subfertile or the normal control bulls. The spermatozoa of one of the 2 subfertile bulls was shown to have a decreased ability to fertilize superovulated ova, while the other subfertile animal, the bull with the lowest noreturn rate, was found by chromosome analysis to have a reciprocal translocation (60, XY, rcp 20:24), causing embryonic death. We suggest that subfertile bulls should not be used in commercial embryo transfer programs nor in artificial insemination and that andrological studies on subfertile bulls with good sperm motility should include evaluation of 6- to 7-day-old ova from superovulated cows to determine if the fertilization rate is normal or impaired. A chromosome analysis should also be performed when a subjertile bull has a normal fertilization rate of ova.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: II. A fertility assay for frozen-thawed semen

Frozen-thawed sperm from five bulls with fertility rates ranging from 48% to 77% were treated with seven concentrations of dilauroylphosphatidylcholine (PC12) liposomes to induce an acrosome reaction (AR) that enabled sperm to penetrate eggs. Treated sperm were incubated with liposomes for 7 min prior to insemination of zona-free hamster eggs in vitro. Sperm and eggs were incubated 3 hr at 39°C prior to fixation, staining, and examination for sperm penetration and nuclear decondensation. The percentage of motile sperm immediately after thawing as well as after treatment with liposomes had a low correlation with sire fertility (r = .39 and ?.63, respectively). The percentage of sperm exhibiting an AR was more highly correlated with fertility (r ? ?.85). Similar correlations were found between fertility and the penetration rates of zona-free hamster eggs or the total number of penetrating sperm. When data for two high and for two lower fertility buils were each grouped to increase information per data point the correlation between the PC12 concentration giving the maximum proportion of eggs penetrated and fertility was r = .92 (P ≤ .05). The correlation between the PC12 concentration producing the most total sperm penetrating the eggs and fertility r = .97 (P ≤ .05). It was concluded that PC12 liposomes induced an AR in bull sperm frozen-thawed in egg yolk extender. Frozen-thawed sperm from low fertility bulls require less PC12 to induce the AR and to penetrate zona-free hamster eggs than do sperm from higher fertility bulls. These differences in lipid requirements may help to provide a quick, direct laboratory assay method to estimate the fertility of frozen bull semen.     

Mapping of insertion elements IS1, IS2 and IS3 on the Escherichia coli K-12 chromosome. Role of the insertion elements in formation of Hfrs and F' factors and in rearrangement of bacterial chromosomes

The chromosome of an Escherichia coli K-12 strain W3110 contains seven copies of insertion element IS1, 12 copies of IS2 and six copies of IS3. We determined the approximate locations of six copies of IS1 (named is1A to is1F), ten copies of IS2 (named is2A to is2J), and five copies of IS3 (named is3A to is3E) on the W3110 chromosome by plaque hybridization using the "mini-set" of the lambda phage library that includes 476 clones carrying chromosomal segments that cover the W3110 chromosome almost entirely. Cleavage maps of the W3110 chromosome and cleavage analysis of phage DNAs carrying insertion elements allowed us to assign more precise locations to most of the insertion elements and to determine their orientations. Insertion elements were distributed randomly along the W3110 chromosome in one or other orientation. Several of these were located at the same positions on the chromosome of another E. coli K-12 strain, JE5519, and they were assumed to be the original complement of insertion elements in E. coli K-12 wild-type. Locations and orientations of such insertion elements were correlated well with Hfr points of origin and with crossover points for excision of some F' factors derived from several Hfrs. Insertion elements may be involved also in rearrangement of bacterial chromosomes.     

Fertility-associated proteins in Nelore bull sperm membranes

The present study was undertaken to evaluate the protein composition of the sperm membranes (SM) of Nelore bulls, assessing protein markers associated with bull fertility, and whether these markers can be used for predicting bull fertility. Samples were obtained of 20 Nelore bulls, with fertility ranked and divided into three groups (greater, normal and least). To rank the bull's fertility weighted classification was used (according to the number of pregnant cows, number of AI cows and number of herds, considering three different breeding seasons), using the PROC GENMOD as a statistical model, with 99% significance. A total of 7897 Nelore cows, randomly distributed among 28 different farms, were considered in the statistical analyses. The bulls were divided into three fertility groups (pregnancy rates): greater (%F > 80), normal (79 < %F > 71) and least (< 68%F) with 3, 13 and 4 bulls, respectively. Two-dimensional gel electrophoresis (2DE) of sperm membranes indicated in 27 spots (SM40, SM53, SM69, SM93, SM102, SM111, SM137, SM138, SM189, SM196, SM201, SM202, SM204, SM225, SM236, SM237, SM239, SM241, SM246, SM247, SM275, SM283, SM342, SM346, SM355, SM372, SM391) was prevalent in the higher fertility group, and just one spot (SM244) was prevalent in the lower fertility group. Spots SM244 and SM239 had their identification defined by PMF/MALDI-MS, as BSP-A3 and aSFP, respectively. Both these proteins showed a great potential for predicting bull's fertility. The amount of aSFP was 8.5 times greater in the sperm membrane protein profile of the higher fertility groups of Nelore bulls. Besides that, the BSP-A3 was 2.5 times greater in the lower fertility group. For the other spots potentially associated with fertility not yet identified, additional tests will be necessary, but it is clear that the 2D electrophoresis of the sperm membrane can be used for a new approach to predict Nelore bull fertility.     

A quantitative trait locus for live weight maps to bovine Chromosome 23

A multiple-marker mapping approach was used to search for quantitative trait loci (QTLs) affecting production, health, and fertility traits in Finnish Ayrshire dairy cattle. As part of a whole-genome scan, altogether 469 bulls were genotyped for six microsatellite loci in 12 families on Chromosome (Chr) 23. Both multiple-marker interval mapping with regression and maximum-likelihood methods were applied with a granddaughter design. Eighteen traits, belonging to 11 trait groups, were included in the analysis. One QTL exceeded experiment level and one QTL genome level significance thresholds. Across-families analysis provided strong evidence (P_experiment= 0.0314) for a QTL affecting live weight. The QTL for live weight maps between markers BM1258 and BoLA DRBP1. A QTL significant at genome level (P_genome= 0.0087) was mapped for veterinary treatment, and the putative QTL probably affects susceptibility to milk fever or ketosis. In addition, three traits exceeded the chromosome 5% significance threshold: protein percentage of milk, calf mortality (sire), and milking speed. In within-family analyses, protein percentage was associated with markers in one family (LOD score = 4.5). Received: 14 December 1998 / Accepted: 28 March 1998     

Serum testosterone concentration in response to exogenous GnRH does not predict service rates or pregnancy rates by yearling, performance tested, crossbred bulls

Ten bulls with a scrotal circumference of less than 30 cm at the end of growth performance testing, and 10 cohorts of the same age, size and breed type with a scrotal circumference greater than 30 cm were used to evaluate if testosterone response following GnRH administration could be used to test for fertility, for semen quality, and for specific pathologic testicular parenchymal changes. Serum testosterone concentrations were determined immediately before and 2 to 3 hours following intramuscular injection of 250 ug GnRH. Bulls were examined for breeding soundness, then fertility was tested in a breeding trial; testicular histology was assessed by determining the percentage of cross-sections of seminiferous tubules with no spermatocytes. The mean (+/- SEM) post-GnRH serum testosterone concentration for all bulls was 11.71 (+/-0.64) ng/ml. In order to examine for an association, the GnRH response was classified as above or below the mean for resultant serum testosterone concentration. The GnRH response classification was not related to the scrotal circumference, percentage of tubules devoid of spermatocytes, or percentage of progressively motile spermatozoa (P > 0.10). The percentage of morphologically normal spermatozoa was significantly higher (P < 0.05) in the bulls with a higher than mean testosterone secretion in response to GnRH injection. In the breeding trial, the percentage of heifers bred and the percentage of heifers pregnant (60 days post breeding) were not significantly different (P > 0.10) between the 2 classifications of GnRH response. The GnRH response test was related to the percentage of morphologically normal spermatozoa but did not predict fertility of yearling bulls in this study.