BLAD and CVM are Holstein-specific recessive mutations which have had significant economic impact on dairy cattle breeding worldwide. The aim of this study was to optimize a diagnostic test for detection of these two disorders and to obtain a preliminary picture about theire existence in Macedonian HF population. Blood samples were obtained from 84 HF cows from several different farms in Macedonia. Genomic DNA was extracted using DNeasy blood and tissue kit (Qiagen) which was also used for DNA extraction from deeply frozen sperm doses from 6 different HF bulls. Genotyping was performed by PCR-RFLP analysis with TaqI and HaeIII restriction endonucleases for BLAD and with PstI for CVM. The correctness of the genotyping was evaluated by direct sequencing of the amplified products. Among 90 HF cattle tested two cows were identified as carriers of the BLAD allele and one was carrier of the CVM allele. No bulls were found to be carriers of either of the two disorders. This study demonstrates that carriers of BLAD and CVM are present in Macedonia, although at low frequency. PCR followed by RFLP is efficient and inexpensive method for detection of the carriers and should be used as laboratory test in eradication programs. In order to establish a more accurate picture of the prevalence of these disorders, it is necessary to screen a larger number of animals. It is indispensable to test each bull before entering the A.I. program to prevent accidental transmission to a large number of offspring.
The objective of the present study was to determine the prevalence of postpartum dysgalactia syndrome (PDS) and associated clinical signs in farmed sows in the Republic of Macedonia (RM) in the first 12-24 h postpartum. A total of 202 sows of different parity and different genetic lines from 5 pig farms in RM were included in the study. The sows and their litters were clinically examined 12-24 hours after farrowing. Postpartum dysgalactia syndrome was detected in 23.3% of all clinically examined sows, while prevalence between farms ranged from 14.8% to 38.1%. Altered piglet’s behavior was the most frequent clinical pattern observed in 68.1% of the PDS–affected (PDSA) sows. Regarding the clinical signs in PDSA sows detected among farms, significant differences were observed in the altered piglet’s behavior (p<0.05) and hypogalactia (p<0.05). Endometritis was more often detected in older sows (90%) compared to endometritis in younger animals (44.4%). In addition, fever was also more frequently diagnosed in higher parity (≥3 parity) sows (55.0%) in contrast to other PDSA sows (22.2%). This study has demonstrated the presence of PDS in farmed sows in RM. High frequency of altered piglet’s behavior found in this study could be an useful indicator for early detection of lactation problems in sows. Frequent pathological vaginal discharge in older sows indicates that endometritis plays an important role in the clinical manifestation of PDS. Further investigations should be conducted in order to identify specific risk factors associated with clinical PDS in farmed sows in RM.
The bovine kappa-casein (κ-CN) is a phospho-protein with 169 amino acids encoded by the CSN3 gene. The two most common gene variants in the HF breed are CSN3*A and CSN3*B while CSN3*E has been found with lower frequency. The aim of this study was to optimize a laboratory method for genotyping of these three alleles as well as to determine their genotype and allele frequencies in the HF cattle population in the Republic of North Macedonia. Genomic DNA was extracted from full blood from 250 cows. The target DNA sequence was amplified with newly designed pair of primers and the products were subjected to enzymatic restriction with HindIII and HaeIII endonucleases. Genotype determination was achieved in all animals. The primers successfully amplified a fragment of 458 bp and the digestion of this fragment with both endonucleases enabled differentiation of five different genotypes with the following observed frequencies: AA (0.39), AB (0.29), BB (0.16), AE (0.10), and BE (0.06). The estimated allele frequencies were: CSN3*A (0.584), CSN3*B (0.336) and CSN3*E (0.08). The observed genotype frequencies differed significantly (P<0.01) from those that would be expected under HW equilibrium, while the fixation index (F=0.17) indicated moderate heterozygosity deficiency. Nevertheless, the CSN3*B allele was present with relatively high frequency which should be used to positively select for its carriers, since increasing its frequency could help to improve the rheological properties of the milk intended for cheese production.
The objectives of the present study were to examine the fatty acid (FA) profiles in serum and in the follicular fluid (FF) and the association between polyunsaturated fatty acid level (PUFA) and follicular growth dynamics following induced luteolysis in dairy cows. A total of 29 dairy cows (CL>25mm, follicle≈15mm) at d0 (start of the experiment) were submitted to ultrasound guided transvaginal follicular aspiration for FF collection from the largest follicle and were injected with 500 μg of cloprostenol. The cows were subdivided into Group A1 (n=11) and Group A2 (n=8) resuming follicular growth either from a secondary follicle less than or larger than 8.5mm, respectively, present at the moment of aspiration and Group A0 (n=10) not resuming follicular growth. Follicular development was monitored daily by ultrasonography until the next dominant follicle reached ≈15mm and was subsequently punctured in Group A1 and A2 (d1). Serum and FF samples for FA determination were taken at d0 from all cows and at d1 in Group A1 and A2. No differences were observed between the FA profile in serum nor in FF between sampling days. Regarding the PUFA levels, the serum linoleic acid (C18:2n6) levels at d0 and d1 were significantly higher than in FF, while alpha linolenic acid (C18:3n3) was lower in the serum than in FF, both at d0 and d1. At d0, a tendency for negative correlation between serum and the FF C18:2n6 with subsequent daily follicular growth rate was observed, while, at d1 there was a strong negative correlation between the serum C18:2n6 and daily growth rate (r=−0.71; p=0.0006). The present study revealed similarities of the FA profiles in the serum and in the FF and association between serum and FF PUFA content with the follicular dynamics after induced luteolysis.
Reduced glutathione (GSH) and homologous ram seminal plasma (HSP), used as additives in cryopreserving (CP) media prior to freezing, showed conflicting results in retaining structural integrity and progressive motility in post-thawed ram spermatozoa. The aims of this research were: (1) to assess the effect of GSH and/or HSP supplementation via soybean-lecithin CP extender on cryopreserved ram spermatozoa viability, morphology and motility pattern; and (2) to assess the effect of incubation in the context of the previous aim. Quantitatively and qualitatively, homogenized and pooled ram ejaculates (N=10) were extended with one of the following extenders: control (C) – tris-based, GSH and HSP-free, experimental-1 (E1) – C + GSH 5 mM, experimental-2 (E2) – C + HSP 20 % and experimental-3 (E3) - GSH 5 mM + HSP 20 %. Following thawing, samples were taken at 0- and 3-hours from each group (n=10) and were assessed for spermatozoa viability, morphology, and motility pattern. C-0h samples yielded a spermatozoa population with low viability, altered head morphology and highly deviated motility pattern. E3-3h samples yielded spermatozoa with unaffected viability, head morphology and high progressive motility. In conclusion, E3 extender added to cryopreserved-thawed ram spermatozoa is most efficient in obtaining high viability, unaltered head morphology, and progressive motility.
The present study aimed to evaluate whether the induction and the formation of an accessory corpus luteum (CL) after AI might increase the pregnancy per AI (P/AI) in heat stressed dairy cows. Starting at d 50±3 post-partum, 113 lactating Holstein cows from one commercial herd during summer were scored for body condition, blood sampled and examined by ultrasound. Those bearing a CL>25mm and progesterone (P4) level>2ng/mL were synchronized using a double PGF2α injection given 12 h apart and AI-ed at detected estrus. In total 18 cows, there were not any signs of estrus (n=10) nor a P4 level <2ng/mL at the time of enrolment (n=8) and therefore they were excluded from the study, leading to 95 cows finally enrolled.. At d5 post-AI, cows were randomly allocated into 2 groups: control group (CON, n=45) without any additional treatment, and treatment group (GnRH, n=50), treated with 0.008 mg Buserelin – a GnRH agonist. Blood sampling and ultrasound examination were done at d5, d14 and at d21 after AI, whereas the pregnancy diagnosis was done at d21 and d30 after AI. Average daily temperature and relative humidity values were used to calculate the temperature-humidity index (THI). The average THI during the experiment was 79.5±0.6. At d5, no differences were observed neither between the number of the CL nor between the P4 level in both groups. At d14 and d21, 82% of the GnRH-treated cows had more than one CL versus 0% of the CON cows. Both at d14 and 21, GnRH-treated cows had higher P4 levels compared to the CON cows (p<0.05). In addition, P/AI were higher in the GnRH group than in the CON group (65% vs. 48.3%, p<0.05), whereas late embryonic losses were higher in CON in comparison to GnRH cows (10.6 vs. 4%, respectively). The BCS at the moment of insemination did not affect P/AI (p>0.05). In conclusion, the induction of an accessory CL at d 5 after AI might increase P/AI in heat stressed dairy cows.
We hypothesized that a single dose of PGF2α belatedly injected on day 8 after GnRH-1 in cows receiving a 7-day Ovsynch-56 protocol (GnRH – 7 days – PGF2α – 56h – GnRH – 16h – timed AI) will increase the proportion of cows with complete luteolysis. At day 35±3 postpartum, 70 lactating Holstein cows from one herd were scored for body condition and pre-synchronized with PGF2α and GnRH (3 days apart) and 7 days later submitted to an Ovsynch-56 protocol for first AI after random assignment to two treatments: (1) OV-7 (n=35) with an injection of PGF2α either on day 7; or (2) OV-8 (n=35) on day 8 after G1, respectively. Blood was collected before the first PGF2α, at day 7 and day 8 in OV-7 and OV-8, respectively, at AI and at 7 days after AI to assess progesterone concentration. Ten cows were classified as acyclic and were excluded from the analysis resulting in 60 cows (OV-8, n=27; OV-7, n=33). In total, more (P=0.01) OV-8 cows and more (P=0.04) primiparous OV-8 cows had complete luteolysis compared with their OV-7 herd mates. In addition, more (P=0.008) OV-8 cows with BCS<2.75 had complete luteolysis compared with their OV-7 herd mates, whereas no difference was observed between treatments among cows with BCS ≥2.75. In conclusion, delaying the application of PGF2α by 1 day reduced the percentage of primiparous cows and cows with poorer BCS having incomplete luteal regression at the time of AI.