Scrotal insulation and its relationship to abnormal morphology, chromatin protamination and nuclear shape of spermatozoa in Holstein-Friesian and Belgian Blue bulls

The objectives of this study were to identify the stages of spermatogenesis susceptible to elevated testicular temperature in terms of sperm motility, viability, morphology, chromatin protamination and nuclear shape. The latter two valuable parameters are not included in routine semen analysis. Scrotal insulation (SI) was applied for 48 h in 2 Holstein-Friesian (HF) and 2 Belgian Blue (BB) bulls and semen was collected at 7 d intervals along with semen collection of a non-insulated bull of each breed. Semen samples were frozen and assigned to 4 groups: period 1 (preinsulation) = −7 d and 0 d, where 0 d = initiation of SI after semen collection; period 2 = 7 d (sperm presumed in the epididymis during SI); period 3 = 14 d to 42 d (cells presumed at spermiogenesis and meiosis stages during SI); period 4 = 49 d to 63 d (cells presumed at spermatocytogenesis stage during SI). The percentages of progressively motile and viable spermatozoa as assessed by computer-assisted sperm analysis (CASA) and fluorescence microscopy, respectively were decreased whereas abnormal sperm heads, nuclear vacuoles and tail defects were increased at period 3 (P < 0.05) compared to period 1, 2 or 4 in SI bulls of both HF and BB breeds. Protamine deficient spermatozoa as observed by chromomycin A₃ (CMA₃) staining were more present (P < 0.05) at period 2 and 3 in both breeds compared to period 1 or 4. Sperm nuclear shape as determined by Fourier harmonic amplitude (FHA) was most affected by heat stress during period 3 (P < 0.01) and a higher response was observed in BB bulls than HF bulls. In conclusion, sperm cells at the spermiogenic and meiotic stages of development are more susceptible to heat stress. The lack of chromatin protamination is the most pertinent result of heat stress, together with subtle changes in sperm head shape, which can be detected by FHA but not by conventional semen analysis.     

Effect of Vitamin E and Polyunsaturated Fatty Acids on Cryopreserved Sperm Quality in Bos taurus Bulls Under Testicular Heat Stress

Taurine bulls are highly susceptible to heat stress, leading to increased oxidative stress (OS) and impaired sperm viability. Polyunsaturated fatty acids (PUFAs) supplementation can be an alternative to improve semen quality, which also results in more sperm susceptibility to lipid peroxidation. Moreover, this deleterious effect can be exacerbated in animals affected by heat stress. Vitamin E is a key antioxidant that counteracts lipid peroxidation of sperm membrane caused by OS. Thus, combining PUFAs with vitamin E may improve sperm quality. In this context, this study aimed to evaluate the effect of interaction between PUFAs and vitamin E on sperm quality in Bos taurus bulls under testicular heat stress. Sixteen taurine bulls under testicular heat stress were randomly assigned in four groups: Control, Vitamin E, PUFA, and PUFA?+?Vitamin E. All groups lasted for 60 days. Samples were cryopreserved/thawed and analyzed for motility variables (CASA), membrane and acrosome integrity, mitochondrial activity, susceptibility to oxidative stress, DNA integrity, and sperm-binding capacity. Results showed that vitamin E had a beneficial effect on some sperm characteristics, whereas PUFA supplementation had an adverse effect when the two treatments were evaluated separately. Finally, the association between PUFAs and vitamin E did not improve sperm quality.     

Sexual development in early- and late-maturing Bos indicus and Bos indicus x Bos taurus crossbred bulls in Brazil

Two experiments were conducted to evaluate sexual development in early- and late-maturing Nelore (Bos indicus) and Canchim (3/8 Bos indicus x 5/8 Bos taurus crossbred) bulls and to determine predictors of sexual precocity, and pubertal and maturity status. In Experiment 1, 12 Nelore bulls where examined from 300 to 900 days of age. Puberty was characterized by an ejaculate containing > or =50 million sperm with > or =10% motile sperm, and maturity by an ejaculate containing > or =70% morphologically normal sperm. In Experiment 2, 28 Canchim bulls where examined from 295 to 488 days of age and puberty was characterized by an ejaculate containing > or =30% motile sperm. In both experiments, bulls were classified as early- or late-maturing based on age at puberty. Early-maturing bulls were younger (P < 0.05) than late-maturing bulls at puberty (527 days versus 673 days in Experiment 1 and 360 days versus 461 days in Experiment 2) and at maturity (660 days versus 768 days in Experiment 1). In general, early-maturing bulls were heavier and had greater scrotal circumference (SC), testes, and testicular vascular cone diameter than late-maturing bulls during the experimental period. Scrotal circumference adjusted for 365 days of age was a good predictor of sexual precocity; minimum yearling SC of 19 and 24 cm for Nelore and Canchim bulls, respectively, had the best predictive values. Early-maturing bulls were lighter and had smaller SC at puberty than late-maturing bulls; therefore, sexual precocity was not related to the attainment of a threshold body weight or testicular size earlier, but to lower thresholds in early-maturing bulls. When predictors of pubertal status were evaluated, SC had the best sensitivity/specificity relationship in Nelore bulls, and high sensitivity and specificity in Canchim bulls. When predictors of sexual maturity were evaluated in Nelore bulls, age, weight, and SC had similar sensitivity, specificity, and predictive values. At puberty, approximately 60% of the sperm present in the ejaculate were morphologically defective. Changes in semen quality after puberty in Nelore bulls were characterized by increased motility and proportion of morphologically normal sperm, with a decrease in the proportion of major sperm defects. In conclusion, early-maturing bulls were more developed in the pre-pubertal period and attained puberty at earlier stages of body and testicular development than late-maturing bulls. Yearling SC could be used to select bulls for sexual precocity and SC was the best predictor of pubertal status. Age, weight, and SC were equally good predictors of sexual maturity in B. indicus bulls.     

Effects of zeranol upon bovine testicular function

Effects of zeranol upon testicular function in bulls were studied. In Experiment I, young bulls (104+/-1.7 days of age) were either implanted with 36 mg zeranol or served as controls with reimplantation on days 56 and 112 after first implant. After a 168-day treatment period, half the bulls were orchidectomized with the remaining bulls assigned to a 168-day recovery period, after which they were also orchidectomized. Zeranol inhibited testicular development during the treatment period. Testosterone (T) concentrations were lower (P<0.01) in treated bulls. Spermatozoa were absent in testes of treated bulls while control bulls averaged 27x10(6) sperm per gram of testis. At the end of the 168-day recovery period, testes weights and sperm concentrations were similar (P>0.10) between bulls previously treated with zeranol and controls. Implantation of older bulls with either 36 mg (267+/-3.4 days of age: Experiment II) or 72 mg (16 months of age: Experiment III) of zeranol had no effect (P>0.10) upon spermatogenesis or T production over a 168-day treatment period. The results indicate that the inhibitory effects of zeranol upon bovine testicular function are age-dependent and suggest that there may be an increased sensitivity of the hypothalmic-pituitary-testicular axis to zeranol in the prepuberal bull.     

Spectral Libraries for SWATH‐MS Assays for Drosophila melanogaster and Solanum lycopersicum

Quantitative proteomics methods have emerged as powerful tools for measuring protein expression changes at the proteome level. Using MS‐based approaches, it is now possible to routinely quantify thousands of proteins. However, prefractionation of the samples at the protein or peptide level is usually necessary to go deep into the proteome, increasing both MS analysis time and technical variability. Recently, a new MS acquisition method named SWATH is introduced with the potential to provide good coverage of the proteome as well as a good measurement precision without prior sample fractionation. In contrast to shotgun‐based MS however, a library containing experimental acquired spectra is necessary for the bioinformatics analysis of SWATH data. In this study, spectral libraries for two widely used models are built to study crop ripening or animal embryogenesis, Solanum lycopersicum (tomato) and Drosophila melanogaster, respectively. The spectral libraries comprise fragments for 5197 and 6040 proteins for S. lycopersicum and D. melanogaster, respectively, and allow reproducible quantification for thousands of peptides per MS analysis. The spectral libraries and all MS data are available in the MassIVE repository with the dataset identifiers MSV000081074 and MSV000081075 and the PRIDE repository with the dataset identifiers PXD006493 and PXD006495.     

Effects of scrotal insulation on sperm production,semen quality,and testicular echotexture in Bos indicus and Bos indicus x Bos taurus bulls

The objectives of the present study were to evaluate the effects of scrotal insulation on sperm production, semen quality, and testicular echotexture in Bos indicus and Bos indicus x Bos taurus crossbred bulls. In one experiment, B. indicus bulls (n=12) were allocated to control and whole-scrotum insulation groups, while in a second experiment, crossbred bulls (n=21) were allocated into control, whole-scrotum, and scrotal-neck insulation groups. Insulation was applied for 4 days (start of insulation = Day 0) and semen collection and testicular ultrasonographic examinations were performed twice weekly until Day 35. Sperm concentration and total sperm output during the post-insulation period were greater in control groups, but significant differences were observed only in B. indicus bulls. Overall, sperm motility in scrotal-insulated B. indicus bulls was lower (P<0.05) than in the control group. After whole-scrotum insulation in crossbred bulls, sperm motility was lower (P<0.05) than pre-insulation levels between Days 21 and 31, and lower than control levels on Day 24. The proportion of normal sperm after whole-scrotum insulation was lower than pre-insulation and control values from Day 11 to the end of the experiment in B. indicus bulls (P<0.05 from Days 14 to 21 and on Day 27), and from Days 14 to 25 in crossbred bulls (P<0.05 on Days 14 and 18). Insulation of the scrotal neck in crossbred bulls did not significantly affect semen quality. Loose sperm heads (Day 11), midpiece defects (Days 11 and 14), and acrosome defects (Days 27 and 31) increased (P<0.05) in insulated B. indicus bulls, while proximal cytoplasmic droplets (Days 14, 18 and 27 in B. indicus; Days 24 and 27 in crossbred bulls) and sperm vacuoles (Days 18 and 21 in B. indicus; Day 18 in crossbred bulls) increased (P<0.05) in whole-scrotum insulation groups in both experiments. There was considerable variation among bulls in the incidence of specific sperm defects. The timing of appearance of sperm defects after insulation provided insights into the pathogenesis of specific abnormalities. Neither whole-scrotum nor scrotal-neck insulation affected testicular echotexture in either experiment. In conclusion, whole-scrotum insulation resulted in decreased sperm production and semen quality in B. indicus and B. indicus x B. taurus bulls, but those changes were not associated with changes in testicular echotexture.     

Testicular thermoregulation in Bos indicus, crossbred and Bos taurus bulls: relationship with scrotal, testicular vascular cone and testicular morphology, and effects on semen quality and sperm production

Mechanisms of testicular thermoregulation, the relationship of scrotal, testicular vascular cone (TVC), and testicular morphology with thermoregulatory capability, and their effects on semen quality and sperm production were studied in 20 Bos indicus, 28 crossbred, and 26 Bos taurus bulls. The ratio of testicular artery length and volume to testicular volume were larger (P<0.05) in B. indicus and crossbred bulls than in B. taurus bulls (1.03 and 0.94 cm/cm3 versus 0.48 cm/cm3; 0.034 and 0.047 ml/cm3 versus 0.017 ml/cm3, respectively). Testicular artery wall thickness (average 192.5, 229.0, and 290.0 microm, respectively) and arterial-venous blood distance in the TVC (average 330.5, 373.7, and 609.4 microm, respectively) were smallest in B. indicus, intermediary in crossbred, and greatest in B. taurus bulls (P<0.05); the proximity between arterial and venous blood was consistent with the estimated decrease in arterial blood temperature after passage through the TVC (5.9, 5.0, and 2.9 degrees C, in B. indicus, crossbred, and B. taurus bulls, respectively). In crossbred and B. taurus bulls, there was a positive top-to-bottom scrotal temperature gradient and a negative testicular subtunic temperature gradient. However, in B. indicus bulls, both scrotal and testicular subtunic temperatures gradients were positive. Differences in the vascular arrangement, characteristics of the artery (e.g. wall thickness) or thickness of the tunica albuginea may have affected the testicular arterial blood and subtunic temperatures in B. indicus bulls. Better testicular thermoregulatory capability was associated with increased scrotal shape (pendulosity), testicular artery length and volume, and top-to-bottom gradient of the distance between the artery wall and the veins in the TVC. Increased semen quality was associated with increased testicular volume and scrotal subcutaneous (SQT) temperature gradient, and with decreased scrotal surface and testicular temperatures. Increased sperm production was associated with increased testicular artery volume, testicular volume, and SQT temperature gradient, and with decreased testicular artery wall thickness, scrotal circumference (SC), and scrotal surface, testicular subtunic, and epididymal temperatures. In conclusion, morphology of the TVC may contribute to the greater resistance of B. indicus bulls to high ambient temperatures by conferring a better testicular blood supply and by facilitating heat transfer between the testicular artery and veins. Testicular thermoregulation was associated with opposing scrotal and testicular subtunic temperatures gradients only in crossbred and B. taurus bulls. Scrotal, TVC, and testicular morphology influence testicular thermoregulatory capability and were associated with differences in semen quality and sperm production.     

Characterization of seminal plasma proteins and sperm proteins in ejaculates from normospermic bulls and bulls with thermally-induced testicular degeneration

Semen was collected from 5 mature beef bulls by electroejaculation before, during, and after 20 days of scrotal insulation. Thermally-induced testicular degeneration was irreversible in 3 of the bulls. Analysis of sperm and seminal plasma polypeptides revealed that 15 to 30 sperm polypeptides and 25 to 30 seminal plasma polypeptides were indistinguishable between bulls prior to the insulation treatment. Changes in the sperm polypeptides pattern appeared as early as 2 days after the insulation treatment and persisted for at least 11 months in 2 of the bulls. In the spermatozoa, there was a detectable loss of 31, 34, 49 and 58 kDa polypeptides and an appearance of 6 to 8 new major polypeptides, ranging from 32 to 83 kDa. The 83 kDa polypeptide was most prominent in the 2 bulls that regained normal sperm motility and morphology following the insulation period. The post-insulation appearance of a seminal plasma polypeptide (circa 60 kDa) was also identified in these 2 bulls. Seminal plasma polypeptides remained qualitatively unaltered by the insulation treatment in the 3 bulls with irreversible testicular degeneration.     

Testosterone, luteinizing hormone, follicle stimulating hormone, and prolactin response to unilateral castration in prepubertal Holstein bulls

Hemicastration of Holstein bulls at 3 months of age resulted in increased (P<0.005) testicular weitht and testis sperm cell content at 330 days after treatment, but did not alter sperm cell concentration in the remaining hypertrophied testis. Radioimmuroassay of blood hormones at 1, 6, 12, and 24 weeks after treatment revealed that unilateral castration did not alter (P>0.1) basal levels or GnRH response profiles of either LH or testosterone compared to intact bulls. Hemicastration caused FSH to be elevated (P<0.01) compared to intact bulls at all sampling periods in both unstimulated and GnRH stimulated bulls. Prolactin varied with season and was greater (P<0.001) in hemicastrated bulls than in intact bulls at 1 and 6 weeks after treatment. Results indicate that unilateral castration at 3 months of age caused testicular hypertrophy of both steroidogenic and gametogenic function and this phenomena may be triggered by increased FSH or prolactin secretion, or both. Further, results indicate different testicular regulation mechanisms exist for pituitary LH and FSH release in bulls.     

Effect of age and genetic group on characteristics of the scrotum,testes and testicular vascular cones,and on sperm production and semen quality in AI bulls in Brazil

The objectives were to determine the effects of age and genetic group on characteristics of the scrotum, testes and testicular vascular cones (TVC), and on sperm production and semen quality in 107 Bos indicus, B. taurus and cross-bred bulls at three artificial insemination (AI) centers in Brazil. In addition, predictors of sperm production and semen quality were identified. In general, scrotal circumference (SC), scrotal shape score, scrotal neck perimeter, and testicular size (length, width and volume) increased (P < 0.05) with age. Although there were no significant differences among genetic groups for SC or testicular size, B. indicus bulls had the least pendulous scrotal shape, the shortest scrotal neck length, and the greatest scrotal neck perimeter (P < 0.05). Fat covering the TVC was thinner (P < 0.05) in bulls < or = 36 months of age and in B. taurus bulls than in older bulls and B. indicus bulls, respectively. Age and genetic group did not affect testicular ultrasonic echotexture. B. indicus bulls tended (P < 0.1) to have the lowest average scrotal surface temperature (SST). In general, ejaculate volume, total number of spermatozoa and number of viable spermatozoa increased (P < 0.05) with age. However, there was no significant effect of age on sperm concentration, motility, major and total defects. The proportion of spermatozoa with minor defects was highest (P < 0.05) in bulls 37-60 months of age. B. indicus bulls had higher (P < 0.01) sperm concentration, total number of spermatozoa and number of viable spermatozoa than B. taurus bulls, with intermediate values for cross-bred bulls. Increased sperm production was associated with increased testicular volume, SC, TVC fat cover, and SST top-to-bottom gradient. Decreased semen quality was associated with increased SC and bottom SST, and decreased scrotal shape, scrotal neck perimeter and vascular cone diameter. In summary, age and genetic group affected the characteristics of the scrotum, testes, and TVC, sperm production and semen quality. In addition, characteristics of the scrotum, testes and TVC were associated with sperm production and semen quality in bulls and could be assessed for breeding soundness evaluation.     

Immunoneutralization of inhibin modifies hormone secretion and sperm production in bulls.

The objective of this study was to examine the role of endogenous inhibin in the regulation of FSH, LH, and testosterone secretion and sperm production in bulls. Bulls were actively immunized against bovine inhibin alpha 1-26 gly-tyr (bINH) conjugated to human alpha globulin (HAG) or HAG alone (controls) and emulsified in Freund's complete adjuvant. Primary immunization was at 14 wk of age, followed by booster immunizations in Freund's incomplete adjuvant at 28, 30, and 34 wk of age. Ten days after each booster immunization, scrotal circumferences and body weights were measured, and blood was sampled for determination of bINH antibody titer. Ten days after the third booster, blood was sampled at 1-h intervals for 8 h to quantify serum concentrations of FSH, LH, and testosterone. After this blood sampling period, bulls were castrated and testicular sperm production was determined. Serum diluted 1:4,000 from bINH-immunized bulls bound 36%, 52%, and 53% of radioiodinated bINH after the first, second, and third boosters, respectively. Serum from controls bound less than 1% radioiodinated bINH. After the third booster, serum concentrations of FSH and testosterone were increased (p less than 0.05) and LH concentrations were decreased (p less than 0.001) in bINH-immunized bulls compared with controls. After the third booster, daily sperm production per gram of testicular parenchyma was increased (p less than 0.05) in bINH-immunized bulls compared with controls. Scrotal circumferences and body weights were similar between treatment groups throughout the experiment. We concluded that inhibin has a role in regulation of secretion of gonadotrophins and testosterone and testicular sperm production, but not testicular growth, in bulls.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls

The expression of proteins in accessory sex gland fluid (AGF) of proven, high use mature Holstein bulls was evaluated. Thirty-seven bulls with documented fertility based on their non-return rates were studied. AGF was obtained by artificial vagina after bulls were surgically equipped with cannulae in the vasa deferentia. Samples of AGF were evaluated by two-dimensional SDS-PAGE, gels stained with Coomassie blue and polypeptide maps analyzed by PDQuest software. A master gel generated by the software representing the best pattern of spots in the AGF polypeptide maps was used as a reference for protein identification. Proteins were identified by Western blots and capillary liquid chromatography-nanoelectrospray ionization tandem-mass spectrometry (CapLC-MS/MS). The product ion spectra were processed using Protein Lynx Global Server 2.1 prior to database search with both PLGS and MASCOT (Matrix Science) software. The entire NCBI database was considered for mass fingerprint matching. An average of 52+/-5 spots was detected in the AGF 2D gels, which corresponded to proteins potentially involved in capacitation (bovine seminal plasma protein-BSP-A1/A2 and A3, BSP 30 kDa, albumin); sperm membrane protection, prevention of oxidative stress, complement-mediated sperm destruction and anti-microbial activity (albumin, clusterin, acidic seminal fluid protein--aSFP, 5'-nucleotidase--5'-NT, phospholipase A2--PLA2); acrosome reaction and sperm-oocyte interaction (PLA2, osteopontin); interaction with the extracellular matrix (tissue inhibitor of metalloproteinase 2, clusterin) and sperm motility (aSFP, spermadhesin Z13, 5'-NT). The 20 spots distinguished in all gels were matched to proteins associated with these functions. Proteins identified by tandem mass spectrometry as ecto-ADP-ribosyltransferase 5 and nucleobindin, never described before in the accessory sex gland secretions, were also detected. In summary, we identified a diverse range of components in the accessory sex gland fluid of a select group of Holstein bulls with documented fertility. Known characteristics of these proteins suggest that they play important roles in sperm physiology after ejaculation.     

Elevated testicular temperature modulates expression patterns of sperm proteins in Holstein bulls

The objective of this study was to determine the effects of elevated testicular temperature on the expression patterns of sperm proteins in bulls. Ejaculates were collected from sexually mature Holstein bulls (n = 6) twice weekly for 10 weeks. Testicular temperature was elevated in all bulls by scrotal insulation for 72 consecutive hours during Week 2. Triton X-100 extracts of sperm proteins were prepared and subjected to SDS-PAGE. Sperm proteins in the 110 kDa range decreased from pre-thermal insult to Day 28 (start of thermal insult = Day 0), concurrent with decreases in sperm concentration, motility, and morphology, and subsequently increased, approaching pre-thermal insult values by Day 40. Based on mass spectrometry, this band was comprised of angiotensin converting enzyme (ACE), Hexokinase-1, and the alpha-4 subunit of Na(+)/K(+)ATPase. Changes in the expression patterns of these proteins were confirmed by immunoblotting, including the use of a custom antibody against the alpha-4 subunit of Na(+)/K(+)ATPase (testis-specific isoform). Furthermore, a 25 kDa sperm protein (identified as tissue inhibitor of metalloproteinase-2; TIMP-2), had a low expression level in pre-thermal insult samples, increased to Day 28 of post-thermal insult, and subsequently decreased to the pre-thermal insult level by the end of the experimental period. In conclusion, we identified proteins that may serve as molecular markers of impaired sperm function due to elevated testicular temperature, with important implications for fertility predictions. To our knowledge, this is the first report of the identification of a testis-specific isoform of Na(+)/K(+)ATPase in bovine sperm.     

Comparison of iTRAQ and SWATH in a clinical study with multiple time points

Background

Advances in mass spectrometry have accelerated biomarker discovery in many areas of medicine. The purpose of this study was to compare two mass spectrometry (MS) methods, isobaric tags for relative and absolute quantitation (iTRAQ) and sequential window acquisition of all theoretical fragment ion spectra (SWATH), for analytical efficiency in biomarker discovery when there are multiple methodological constraints such as limited sample size and several time points for each patient to be analyzed.

Methods

A total of 140 tear samples were collected from 28 glaucoma patients at 5 time points in a glaucoma drug switch study. Samples were analyzed with iTRAQ and SWATH methods using NanoLC-MSTOF mass spectrometry.

Results

We discovered that even though iTRAQ is faster than SWATH with respect to analysis time per sample, it loses in sensitivity, reliability and robustness. While SWATH analysis yielded complete data of 456 proteins in all samples, with iTRAQ we were able to quantify 477 proteins in total but on average only 125 proteins were quantified in a sample. 283 proteins were common in the datasets produced by the two methods. Repeatability of the methods was assessed by calculating percent relative standard deviation (% RSD) between replicate MS analyses: SWATH was more repeatable (56% of proteins?<?20% RSD), compared to iTRAQ (43% of proteins?<?20% RSD). Despite the overall benefits of SWATH, both methods showed less than 1 log fold change difference in the expression of 74% common proteins. In addition, comparison to MS/MS peptide results using 8 isotopically labeled peptide standards, SWATH and iTRAQ showed similar results in terms of accuracy. Moreover, both methods detected similar trends in a longitudinal analysis of protein expression of two known tear biomarkers.

Conclusions

Overall, we conclude that SWATH should be preferred for biomarker discovery studies when analyzing limited volumes of clinical samples collected at multiple time points.

Trial Registeration

The study was approved by the Ethics Committee at Tampere University Hospital and was registered in EU clinical trials register (EudraCT Number: 2010-021039-14).

     

Influence of season of birth on growth and reproductive development of Brahman bulls

Seasonal effects on reproduction are more dramatic in Bos indicus than Bos taurus cattle. This experiment evaluated reproductive development of fall- (n=7) versus spring- (n = 10) born Brahman bulls to determine if season of birth affects reproductive development. Measurements of growth and reproductive development began after weaning and continued at bi-weekly intervals until each bull reached sexual maturity. Different stages of sexual development were classified according to characteristics of the ejaculate and included first sperm in the ejaculate, puberty (> 50 x 10(6) sperm/ejaculate), and sexual maturity (two ejaculates with > 500 = 10(6) sperm/ejaculate). Average daily increases in all measured traits were similar in fall- and spring-born bulls and there were no differences in age, body weight, scrotal circumference, or paired testis volume between groups at first sperm or puberty. However, fall-born bulls were older (P < 0.05) than spring-born bulls at sexual maturity (553 days versus 481 days, respectively) as the interval between puberty and sexual maturity was longer (P < 0.05) in fall- than in spring-born bulls (82 days versus 54 days, respectively). The prolonged interval between puberty and sexual maturity in fall-born calves coincided with a short photoperiod (winter) whereas the short interval between puberty and sexual maturity in spring-born calves coincided with a long photoperiod (summer). In conclusion, season of birth affected sexual development; photoperiod might be involved in regulating testicular function immediately after puberty in Brahman bulls.     

Testicular and epididymal function during the peripuberal period in Brahman bulls receiving various amounts of protein degradable in the rumen

Thirty-nine Brahman bulls with an initial age and weight of 301.7 +/- 4.1 d and 202.7 +/- 4.7 kg, respectively, were randomly allocated to 1 of 2 dietary treatment groups within age, weight and sire in order to study the influence of source of protein and stage of peripuberal period on testicular and epididymal function. In the soybean meal treatment the amount of protein undegradable in the rumen averaged 47%, while it was 72% in the fish meal treatment. The supplements were isocaloric and isonitrogenous. Bulls were electroejaculated, and castrations were performed randomly in a predetermined order when the first ejaculate with the first motile sperm cells (Stage 1), 10 to 25 million (Stage 2), and 50 million or more sperm cells (Stage 3 - puberty) was obtained. Testicular and epididymal traits were analyzed for a single testicle and epididymis. Daily sperm production, daily sperm production per gram of testicular parenchyma, testicular weight and testicular parenchyma weight were not affected by treatment. Bulls receiving fish meal had heavier (P < 0.01) epididymis than soybean meal-fed bulls (6.6 +/- 1.0 vs 3.9 +/- 0.6 g) but similar (P > 0.05) epididymal sperm reserves. Daily sperm production (1 testicle) was 115.2 +/- 0.1, 447.4 +/- 0.1, 792.7 +/- 0.1 million sperm cells, and daily sperm production per gram of testicular parenchyma was 1.5 +/- 0.5, 3.2 +/- 0.6 and 6.4 +/- 0.6 million sperm cells for bulls at Stage 1, 2 and 3, respectively. Sire and amount of undegradable intake protein had significant (P < 0.05) affects on the distribution of epididymal sperm reserves, with soybean meal-fed bulls having the higher proportions of epididymal sperm reserves in the cauda epididymis.     

Synthesis of transferrin and transferrin mRNA in bovine Sertoli cells in culture and in vivo: sequence of partial cDNA clone for bovine transferrin

Techniques were developed for generating enriched cultures of bovine Sertoli cells and indifferent supporting cells (immature Sertoli cells). The [35S]methionine and [35S]sulfate-labeled proteins secreted by cultured cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The electrophoretic pattern of the major Sertoli cell-secreted proteins was distinct from that of the major proteins secreted by cultured peritubular cells (the predominant contaminating cell type). Five major polypeptides ranging in molecular mass from 22 kDa to 77 kDa were resolved by 2D-PAGE in reducing conditions and were assigned numbers for reference purposes. Polypeptides 1 and 2 appeared to be analogous to two rat Sertoli cell-secreted proteins, sulfated glycoprotein-1 and sulfated glycoprotein-2, because of similar molecular mass, isoelectric point, subunit composition, sulfation, and sialation characteristics. Transferrin was detected in conditioned medium by immunoprecipitation using an antibody to bovine serum transferrin. Cultured Sertoli cells isolated from prepubertal bulls secreted higher levels of transferrin than did cells isolated from infant bulls. An 850 bp cDNA corresponding to the 3' portion of bovine transferrin mRNA was cloned and sequenced. Transferrin message was shown to be present in testicular tissue isolated from infant and prepubertal bulls and it increased as bulls matured. Levels of testicular transferrin mRNA were subsequently shown to correlate with daily sperm production in yearling beef bulls.     

Mass Spectrometry Reveals New Insights into the Production of Superoxide Anions and 4‐Hydroxynonenal Adducted Proteins in Human Sperm

The free‐radical theory of male infertility suggests that reactive oxygen species produced by the spermatozoa themselves are a leading cause of sperm dysfunction, including loss of sperm motility. However, the field is overshadowed on several fronts, primarily because: i) the probes used to measure reactive oxygen species (ROS) are imprecise; and ii) many reports suggesting that oxygen radicals are detrimental to sperm function add an exogenous source of ROS. Herein, a more reliable approach to measure superoxide anion production by human spermatozoa based on MS analysis is used. Furthermore, the formation of the lipid‐peroxidation product 4‐hydroxynonenal (4‐HNE) during in vitro incubation using proteomics is also investigated. The data demonstrate that neither superoxide anion nor other free radicals that cause 4‐HNE production are related to the loss of sperm motility during incubation. Interestingly, it appears that many of the 4‐HNE adducted proteins, found within spermatozoa, originate from the prostate. A quantitative SWATH analysis demonstrate that these proteins transiently bind to sperm and are then shed during in vitro incubation. These proteomics‐based findings propose a revised understanding of oxidative stress within the male reproductive tract.     

Proteomic markers of low and high fertility bovine spermatozoa separated by Percoll gradient

In the context of artificial insemination, male fertility is defined as the ability to produce functional spermatozoa able to withstand cryopreservation. We hypothesized that interindividual variations in fertility depend on the proportion of the fully functional sperm population contained in the insemination dose. The objective of this study was to identify protein markers of the fully functional sperm subpopulation. Insemination doses from four high‐fertility (HF) and four low‐fertility (LF) bulls with comparable post‐thaw quality parameters were selected for proteomic analysis using iTRAQ technology. Thawed semen was centrifuged through a Percoll gradient to segregate the motile (high density [HD]) from the immotile (low density [LD]) sperm populations. Sperm proteins were extracted with sodium deoxycholate and four groups were compared: LD and HD spermatozoa from LF and HF bulls. A total of 498 unique proteins were identified and quantified. Comparison of HD spermatozoa from HF and LF bulls revealed that five proteins were significantly more abundant in the HF group (AK8, TPI1, TSPAN8, OAT, and DBIL5) whereas five proteins were more abundant in the LF group (RGS22, ATP5J, CLU, LOC616319, and CCT5). Comparison of LD spermatozoa from HF and LF bulls revealed that four proteins were significantly more abundant in the HF group (IL4I1, CYLC2, OAT, and ARMC3) whereas 15 proteins were significantly more abundant in the LF group (HADHA, HSP90AA1, DNASE1L3, SLC25A20, GPX5, TCP1, HIP1, CLU, G5E622, LOC616319, HSPA2, NUP155, DPY19L2, SPERT, and SERPINE2). DBIL5, TSPAN8, and TPI1 showed potential as putative markers of the fully functional sperm subpopulation.