Holstein Bull Semen Research Articles

Purification of Holstein Bull Semen Paraoxonase 1 (PON1) by Hydrophobic Interaction Chromatography and Investigation of Its Inhibition Kinetics by Heavy Metals

In this study, paraoxonase 1 (PON1; EC 3.1.8.1) was purified from bull semen, and some characteristics of the enzyme were investigated. In vitro inhibition effect of some heavy metals, including Cu(2+), Mn(2+), Cd(2+), Zn(2+), Ni(2+), and Pb(2+), on the activity of the purified enzyme was also investigated. The purification of bull semen PON1 procedure was composed of two steps: ammonium sulfate precipitation and Sepharose-4B-L-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The enzyme, having a specific activity of 288 EU/mg proteins, was purified 22.67-fold with a yield of 89 %. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme showed the presence of a single band with an apparent MW of 66 kDa. The V max and K M values for the paraoxon substrate were determined as 100 EU and 8.0 × 10(-5) M, respectively. The inhibitory effects of different heavy metals on PON1 activity were determined by using the paraoxon as a substrate. The results showed that all the metals, except for Cd(2+), inhibited the PON1 enzyme activity in a concentration-dependent fashion. IC50 values of Cu(2+), Mn(2+), Zn(2+), Ni(2+), and Pb(2+) were found as 2.59 × 10(-3), 1.17 × 10(-3), 42.74 × 10(-3), 99.10 × 10(-3), 48.80 × 10(-3) mM, respectively. Conversely, Cd(2+) increased the bull semen PON1 enzyme activity. The present study has demonstrated that Cu(2+), Mn(2+), Zn(2+), Ni(2+), and Pb(2+) are serious toxic metals, which are able to increase the risk of oxidative stress development and a subsequent decrease of semen quality.

Effects of polymorphisms in the bovine growth differentiation factor 9 gene on sperm quality in Holstein bulls

Members of the transforming growth factor-β (TGFβ) superfamily are critical regulators of germ cell development that act as extracellular ligands of the signal transduction pathways regulating proliferation, differentiation, apoptosis, and other aspects of cell behavior. Growth differentiation factor 9 (GDF9) is a member of the TGFβ superfamily that plays a critical role in ovarian follicular development and ovulation rate in females; however, its role in the testis has not been well elucidated. Therefore, in this study we investigated the effects of GDF9 mutations on the quality of fresh and frozen semen of Holstein bulls. Two reported single nucleotide polymorphisms of GDF9, A485TA and A625C, were analyzed in 129 Holstein bulls. Analysis of variance revealed that the A485T polymorphism had significant effects on the acrosome integrity rate (P < 0.05), whereas the A625T polymorphism was significantly associated with sperm concentration (P < 0.05). In addition, a significant additive effect on sperm concentration was detected for the A485T polymorphism (P < 0.05), whereas the polymorphisms A485TA and A625C had significant dominant effects on acrosome integrity rate and sperm motility in frozen semen, respectively (P < 0.05). This study is the first to show a significant association of GDF9 with sperm quality traits, and the results implied that GDF9 is involved in the initiation or maintenance of spermatogenesis; however, further verification is needed.

Characterization and activity of angiotensin-converting enzyme in Holstein semen

The study was designed to perform immunodetection in spermatozoa and seminal plasma, immunolocalization in spermatozoa, and evaluation of the enzymatic activity of angiotensin-converting enzyme (ACE) in the semen of Holstein bulls. We used ejaculates from five bulls as part of a regular collection of semen. The monoclonal anti-ACE antibody recognized a single protein band with 100kDa in detergent extract prepared from sperm and in seminal plasma. ACE enzymatic activity in sperm was 43.7, 21.3, 45.6, 60.0, and 57.7mU/mL in bulls 1, 2, 3, 4, and 5, respectively, and 0.3, 2.3, 3.0, 2.3, and 2.6mU/mL in seminal plasma of the same bulls, respectively. The average percentages of sperm with acrosome reactions after treatment with heparin were 28.3%, 28.6%, 35.2%, 25.0%, and 32.3%, respectively. These values were higher than the percentages of acrosome reactions in controls and the captopril group (P<0.05), although no difference was seen between the captopril and control groups (P>0.05). After 4h of incubation, motility in the control group (32.9%) was significantly higher than that in the heparin (15.7%) and captopril (12.1%) groups. No difference was found in motility after the capacitation assay in the heparin and captopril groups (P>0.05). In conclusion, ACE was immunologically localized in the acrosome of the spermatozoa of Holstein bull, the specific enzymatic activity of ACE in detergent-extracted spermatozoa and seminal plasma was inhibited by captopril, and this ACE inhibitor reduced the percentage of sperm with progressive motility and acrosome reactions after capacitation in vitro.

SPERM MORPHOLOGICAL ASSESSMENTS OF FRIESIAN HOLSTEIN BULL SEMEN COLLECTED FROM THREE ARTIFICIAL INSEMINATION CENTERS IN INDONESIA

Morphologically abnormal sperm in semen has been associated with the sub fertility and sterilityfor many years. This study assessed the sperm morphology of Friesian Holstein bull semen which wascollected from three Artificial Insemination centers in Indonesia. Total of 22 bulls were used in thisstudy; an ejaculate from each bull was examined. Three to four glass slides were prepared for each bullsample; a drop of semen was placed on each glass slide, smeared, and air-dried. The smeared sampleswere stained with carbolfuchsin-eosin (Williams stain). Morphological abnormality types were recordedfrom total of 500 spermatozoa. Results demonstrated that all 22 bulls had low sperm abnormality(&lt;10%). Pearshaped was the most frequently type of sperm abnormality found in the samples(0.81±0.93%); while detached head was the lowest (0.01±0.04%).

182 MANAGING FACTORS ASSOCIATED WITH PREGNANCY RATE OF HEIFER RECIPIENTS IN LARGE IN VITRO PRODUCED EMBRYO PROGRAMS

Large programs of in vitro embryo production have been developed in Brazil, but the results are very variable. The objective of this study was to evaluate managing factors that influence pregnancy rate of recipients in a large embryo transfer program. Results of 1104 embryo transfers performed from November 2008 to February 2009 by Transgen Desenvolvimento e Produção Agropecuária Ltda (Uberlˆndia, Minas Gerais, Brazil) were evaluated. Embryos produced in vitro were used: 1/2 Holstein/Gir embryos (n = 139) or 3/4 Holstein/Gir embryos (n = 961) produced from donors 1/2 Holstein/Gir and Gir with Holstein bull semen. Only excellent (grade 1) quality morulae (stage 4), early blastocysts (stage 5), mid- blastocysts (stage 6), or expanded blastocysts (stage 7) were assigned for fresh transfer using the International Embryo Transfer Society guideline for grading embryos. The heifer recipients were 1/2 crossbred Nelore/Simmental, in good health condition, 20 to 30 months of age, 330 to 400 kg of weight, and were maintained on pasture (Tifton) with mineral supplementation ad libitum. The recipients were synchronized as follows: Day 0-intravaginal device with 1.0 g of progesterone (PRIMER®, Tecnopec, São Paulo, SP, Brazil) +2 mg of estradiol benzoate (ESTROGIN®, Farmavet, São Paulo, SP, Brazil); Day 5-150 μg of D-Cloprostenol (PRELOBAN® Intervet, São Paulo, SP, Brazil) + 400IU of eCG (FOLLIGON®, Intervet); Day 8-progesterone device removed; Day 9-1 mg of estradiol benzoate. On Day 17, nonsurgical embryo transfers were performed by a trained technician and synchronization was confirmed by presence of a CL. The effects of the embryo breed, the number of transfers in each recipient (the heifers that failed to get pregnant were used in the next program), and the transfer sequence (i.e. 20 embryo transfers were performed per hour, and approximately 100 per day) on the pregnancy rate was analyzed by logistic regression with the LOGISTIC procedure of SAS (SAS Institute Inc., Cary, NC, USA). The breed of embryo (56.9% for 3/4 Holstein/Gir embryos v. 62.6% for 1/2 Holstein/Gir embryos), the number of transfers in each recipient (first: 56.5% v. second: 61.8% v. third: 55.5% v. ≥fourth: 55.0%), and the transfer sequence during the day of the program (first hour: 57.4% v. second hour: 60.0% v. third hour: 58.1% v. ≥fourth hour: 53.8%) did not influence the pregnancy rate. Within the confines of an extensive embryo transfer program with in vitro produced fresh embryos, a large number of embryo transfers can be executed per day without adversely affecting the pregnancy rate if high-quality embryos are used, the transfers are performed by a trained technician, and recipients are in good health condition and synchronized.

Comparison of Holstein bull semen sources on milk traits in Isfahan province in Iran

Abstract. This research provides a comparison of productivity of daughters in Iran sired by bulls from Canada, Europe, Iran, and the United States. Data were collected by the Animal Breeding Center of Iran on pedigree and first lactation performances of 10,192 Holsteins sired by 186 imported (including: 39 European, 73 American, 74 Canadian) and 133 Iranian bulls, which had their first calving in 35 dairy herds in Isfahan province. The results showed that American and European daughters had the highest performances for milk yield. The highest mean for fat yield was related to European daughters. American sires on average had the highest genetic potential for yield traits; however, they had the lowest fat percentage among compared sire groups. Canadian sire group was intermediate relative to American, European and Iranian groups. Higher intercepts and regression coefficients of conversion equations for American sires showed that more returns will be achieved from one genetic point superiority of American sires relative to Canadian sires in the environments of Isfahan province. The Iranian sire group had the lowest predicted transmitting ability means for both milk and fat yields and a negative predicted transmitting ability mean for fat percentage. This study showed that due to low to intermediate predicted transmitting ability correlations between foreign evaluations and the evaluations made by this study, direct selection based on foreign national evaluations would not lead to optimal results for the traits concerned. So, the genetic potential/expression of imported genetic materials needs to be reevaluated by the Animal Breeding Centers of Iran.

Association of Candidate Genes with Production Traits in Korean Dairy Proven and Young Bulls

This study was performed to offer effective basic data for selection and improvement of Korean dairy cattle through identifying distributional properties among candidate genes (bovine butyrophilin, signal transducers and activators of transcription 5a, and prolactin hormone). In this study, polymorphisms of candidate genes were identified and the relationships between loci and production traits of each gene were analyzed using frozen semen of Holstein bulls (19 proven and 77 candidates). In butyrophilin (BTN) locus, polymorphisms information contents (PIC) value of BTN2 (0.372) was higher than those of others (BTN1; 0.155, BTN3; 0.254, BTN4; 0.169). As a result of analysis of genotyping STAT5a, using single stranded conformational polymorphism (SSCP) method and microsatellite locus, PIC values were 0.189 and 0.457, respectively. And PIC value of prolactin hormone gene was 0.176. In the relationships between genotypes and production traits, BTN3 was associated with 305-day production traits (p<0.05). PTAs for B allele were such as 110.43, 88.28 and 75.25 in BTN1, 3, 4 and these values were higher than those of A allele, but in the case of BTN2, A allele with 154.19 was higher than that of B allele. The results obtained from using candidate genes may be used as an useful index for the genetic improvement of dairy cattle population in Korea, and further studies are needed.

Holstein 보증종모우 및 후보종모우의 선천성 장애 유전좌위 검색에 관한 연구

This study was performed to discriminate defective loci by detection of congenital genetic disorder, to offer basic data for selection and improvement of Korean dairy cattle using frozen semen of Holstein bulls(16 proven and 93 candidate). The results obtained were as follows ; By the detection of DUMP(deficiency of uridine monophophate synthase) for 109 Holstein bulls(16 proven and 93 candidate), DUMP carrier was not found in whole animals. Also, it was possible to early detection of DUMP carrier by using PCR-RFLP(AvaⅠ). As the results of detection for BLAD(bovine leukocyte adhesion deficiency), BLAD carrier was not found in 16 proven bulls. But 5 candidtae bulls are discriminated to BLAD carrier, and it could be predicted to transmitted pathway of inherited loci by pedigree identification. Also, when digesting PCR products using restriction enzyme, results from TaqⅠ restriction enzyme were more efficient than that of HaeⅢ. After detection test of citrullinaemia, it was concluded that proven and candidate bulls were not. However, wide range of research and citrullinaemia genotyping should be performed. As a result of this study, the wide and various research should be performed in genetic disease of animal. And in the selection and breeding of animal, the breeding scheme by completely and continuously management of pedigree should be established.

精液の生化學的研究

Samples of the Holstein bull semen obtained by the artificial vagina method were examined for their spermatozoa and seminal fluid contents and the distribution of water and tyresine in each fraction. The amount of spermatozoa was 8.2 per cent of bull semen, and the seminal fluid was then theoretically amounted to 91.8 per cent of the semen. The content of water in semen, spermatozoa, and seminal fluid was 89.94, 81.09 and 90.40 per sent, respectively. The amount of tyrosine per 100g of fresh samples of semen, spermatozoa, and seminal fluid were determined to be 351.2, 445.3, and 441.1mg, respectively and these values were also determined to be 3.49, 2.33, and 4.54 per cent, respectively, in dried samples.