BovineTalk: machine learning for vocalization analysis of dairy cattle under the negative affective state of isolation.

There is a critical need to develop and validate non-invasive animal-based indicators of affective states in livestock species, in order to integrate them into on-farm assessment protocols, potentially via the use of precision livestock farming (PLF) tools. One such promising approach is the use of vocal indicators. The acoustic structure of vocalizations and their functions were extensively studied in important livestock species, such as pigs, horses, poultry, and goats, yet cattle remain understudied in this context to date. Cows were shown to produce two types of vocalizations: low-frequency calls (LF), produced with the mouth closed, or partially closed, for close distance contacts, and open mouth emitted high-frequency calls (HF), produced for long-distance communication, with the latter considered to be largely associated with negative affective states. Moreover, cattle vocalizations were shown to contain information on individuality across a wide range of contexts, both negative and positive. Nowadays, dairy cows are facing a series of negative challenges and stressors in a typical production cycle, making vocalizations during negative affective states of special interest for research. One contribution of this study is providing the largest to date pre-processed (clean from noises) dataset of lactating adult multiparous dairy cows during negative affective states induced by visual isolation challenges. Here, we present two computational frameworks-deep learning based and explainable machine learning based, to classify high and low-frequency cattle calls and individual cow voice recognition. Our models in these two frameworks reached 87.2 and 89.4% accuracy for LF and HF classification, with 68.9 and 72.5% accuracy rates for the cow individual identification, respectively.

Machine learning approaches to predict and detect early-onset of digital dermatitis in dairy cows using sensor data.

The present study aimed to employ machine learning algorithms based on sensor behavior data for (1) early-onset detection of digital dermatitis (DD) and (2) DD prediction in dairy cows. Our machine learning model, which was based on the Tree-Based Pipeline Optimization Tool (TPOT) automatic machine learning method, for DD detection on day 0 of the appearance of the clinical signs has reached an accuracy of 79% on the test set, while the model for the prediction of DD 2 days prior to the appearance of the first clinical signs, which was a combination of K-means and TPOT, has reached an accuracy of 64%. The proposed machine learning models have the potential to help achieve a real-time automated tool for monitoring and diagnosing DD in lactating dairy cows based on sensor data in conventional dairy barn environments. Our results suggest that alterations in behavioral patterns can be used as inputs in an early warning system for herd management in order to detect variances in the health and wellbeing of individual cows.

Infrared thermography as a non-invasive method for evaluating stress in lactating dairy cows during isolation challenges.

The overall objective of the current data report was to evaluate and test the feasibility of using infrared thermography (IRT) as a non-invasive method for measuring stress signs in lactating dairy cows during short negative challenges, such as visual isolation from herd-mates. The study was carried out at the Experimental Farm of the Research and Development Institute for Bovine Romania, on 20 Holstein-Friesian lactating multiparous dairy cows, between August and September 2022. Cows were housed in two identical tied stanchion barns (170/85 cm), and were isolated individually from the herd for 240 min post-morning milking. Our results shown significant (p ≤ 0.05) rises for both orbital and nasal IRT temperatures following the isolation challenge, suggesting that such approaches could represent adequate tools for assessing social stress in cattle. Overall, current results are in accordance with previous studies which validated both eye and nasal regions as IRT thermal windows for studying the effects of painful and negative contexts on stress response in farmed ruminants, while considering the stress-induced hyperthermia as an integral part of the physiological response to negative stimuli, as well as the current limitations that this tool faces.

Kompetitive Allele Specific PCR Genotyping of 89 SNPs in Romanian Spotted and Romanian Brown Cattle Breeds and Their Association with Clinical Mastitis.

Mastitis is the most common production disease in the dairy sector worldwide, its incidence being associated with both cows' exposure to bacteria and the cows' genetic make-up for resistance to pathogens. The objective of our study was to analyse 89 missense SNPs belonging to six genes (CXCR2, CXCL8, TLR4, BRCA1, LTF, BOLA-DRB3), which were found to be associated with genetic resistance or susceptibility to mastitis. A total of 298 cattle (250 Romanian Spotted and 48 Romanian Brown) were genotyped by Kompetitive Allele Specific PCR (KASP) and a chi-squared test was used for genetic association studies with clinical mastitis. A total of 35 SNPs (39.3%) among the selected 89 SNPs were successfully genotyped, of which 31 markers were monomorphic. The polymorphic markers were found in two genes: TLR4 (rs460053411) and BOLA-DRB3 (rs42309897, rs208816121, rs110124025). The polymorphic SNPs with MAF > 5% and call rates > 95% were used for the association study. The results showed that rs110124025 in the BOLA-DRB3 gene was significantly associated with mastitis prevalence (p ≤ 0.05) in both investigated breeds. Current results show that the SNP rs110124025 in the BOLA-DRB3 gene can be used as a candidate genetic marker in selection for mastitis resistance in Romanian dairy cattle.

Genetic polymorphism in European and African sheep breeds reared in Hungary based on 48 SNPs associated with resistance to gastrointestinal parasite infection using KASP-PCR technique.

This pilot study used an alternative and economically efficient technique, the Kompetitive Allele-Specific Polymerase Chain Reaction (KASP-PCR) to examine 48 SNPs from 11 parasite-resistance genes found on 8 chromosomes in 110 animals from five sheep breeds reared in Hungary; Hungarian Tsigai, White Dorper, Dorper, Ile de France, and Hungarian Merino. Allele and genotype frequencies, fixation index, observed heterozygosity, expected heterozygosity, F statistic, and their relationship with the Hardy-Weinberg equilibrium (WHE) and the polymorphic information content (PIC) were determined, followed by principal component analysis (PCA). As much as 32 SNPs out of the 48 initially studied were successfully genotyped. A total of 9 SNPs, 4 SNPs in TLR5, 1 SNP in TLR8, and 4 SNPs in TLR2 genes, were polymorphic. The variable genotype and allele frequency of the TLRs gene indicated genetic variability among the studied sheep breeds, with the Hungarian Merino exhibiting the most polymorphisms, while Dorper was the population with the most SNPs departing from the HWE. According to the PIC value, the rs430457884-TLR2, rs55631273-TLR2, and rs416833129-TLR5 were found to be informative in detecting polymorphisms among individuals within the populations, whereas the rs429546187-TLR5 and rs424975389-TLR5 were found to have a significant influence in clustering the population studied. This study reported a moderate level of genetic variability and that a low to moderate within-breed diversity was maintained in the studied populations.

Validation of SNP markers for thermotolerance adaptation in Ovis aries adapted to different climatic regions using KASP-PCR technique.

A study on 51 SNPs belonging to 29 genes related to heat stress was carried out in 720 sheep from 17 different breeds adapted to different climates from Hungary, Bosnia and Herzegovina, Morocco and Romania, using Kompetitive Allele-Specific Polymerase Chain Reaction. Genotype frequency and the Hardy-Weinberg equilibrium were calculated, followed by a clustering using the Principal Component Analysis. We analyzed the polymorphisms in the following genes analyzed: HSPA12A, HSP90AA1, IL33, DIO2, BTNL2, CSN2, ABCG1, CSN1S1, GHR, HSPA8, STAT3, and HCRT. We emphasized on HSPA12A and HSPA8 genes as they were successfully genotyped in all studied flocks in which genotype frequency patterns were identified. Contrary to previous findings, the A allele for HSPA8 SNP was not observed in the heat tolerant breeds, being found exclusively in cold-tolerant breeds. The principal component analysis could not clearly differentiate the breeds, while plot concentration was slightly varied among the three groups, with HSP90AA1 and IL33 SNPs' loading values significantly contributing to PC1 and PC2. We confirmed previous works that the HSPA12A, HSPA8, HSP90AA1 and IL33 SNPs are potential candidate markers for thermotolerance adaptation in sheep. This research contributes to the genetic variability of SNPs for thermotolerance adaptability in sheep.

Water Buffalo Responsiveness during Milking: Implications for Production Outputs, Reproduction Fitness, and Animal Welfare.

The overall objective of this study was to evaluate the effects that milking temperament (MT) of water buffaloes has on milking traits, welfare indicators, and reproductive outputs. The study was performed on 60 multiparous buffalo cows (6.7 ± 0.6 lactations) at the beginning of their lactation (100 days in milk, DIM). Each buffalo cow was scored by two independent observers using a temperament scoring system (1: extremely calm, 2: calm, 3: alert, 4: reactive, and 5: aggressive), and then grouped as 'calm' (scores 1, 2, and 3; n = 42) or 'nervous' (scores 4 and 5; n = 18). Additionally, the milk yield at 100 DIM (MY), milking speed (MS), calving interval (CI), age at first calving (AFC), body condition score (BCS), animal-based welfare parameters, and infrared thermography data (IRT) were evaluated. The MT significantly influenced the MY (p = 0.0082), with calmer cows outperforming their nervous counterparts. The MS was significantly influenced by the MT (p = 0.0015), with calmer animals having a higher milk ejection rate. The MT of the cows had no influence on the CI, AFC, or BCS. The evidence from this study suggests that the responsiveness of buffalo cows during milking affects their milk yield and milking speed, with no associations being found for reproduction efficiency indicators or animal-based welfare indicators.

Evaluation of single nucleotide polymorphisms identified through the use of SNP assay in Romanian and Chinese Holstein and Simmental cattle breeds.

Simmental and Holstein cattle, being among the most widely distributed breeds worldwide, have been subjected to continuous selection for distinct purposes. In the current study, we evaluated the levels of SNPs identified through the use of SNP assay in Romanian Holstein and Romanian Simmental cattle, which then were compared to the data from Chinese Holstein and Chinese Simmental cattle. In total, 282 animals were genotyped: Romanian Holstein (n=30), Romanian Simmental (n=22), Chinese Holstein (n=96) and Chinese Simmental cattle (n=136), using 39,724 common SNPs to analyze minor allele frequency, genetic variability and level of SNPs. Among studied breeds, the average percentage of polymorphic markers was 90.84%, with the highest value in Chinese Simmental (91.37%) and lowest in Romanian Simmental cattle (90.31%). The average HO ranged from 0.426 in Romanian Holstein to 0.416 in Romanian Simmental, and from 0.425 in Chinese Holstein to 0.422 in Chinese Simmental. The distribution of SNPs was homogenous across the breeds, except the Romanian Simmental which displayed the lowest percentage of polymorphic markers (24,66 and 32,48%) from higher MAF category (0.3 to <0.4 and 0.4 to <0.5) and the highest percentage (3.82 and 12.00%) for SNPs from low and intermediate MAF categories (0.05 to <0.1 and 0.1 to <0.2). In the current study, the SNP assay was successfully used to analyze the level of SNP sites of Romanian cattle breeds, however, a higher number of samples and production data are needed for future applications of the results in genomic selection, genome-wide association studies and genetic diversity analysis.

Genetic characterization of indigenous goat breeds in Romania and Hungary with a special focus on genetic resistance to mastitis and gastrointestinal parasitism based on 40 SNPs.

Goat breeding has become an important sector in Eastern Europe, with Romania and Hungary being among the major producer countries. Given the limited number of research done up-to-date concerning genetic studies of indigenous goat breeds reared in Romania and Hungary, the current preliminary study aimed to analyze the variability of genes related to mastitis and gastrointestinal parasitism by using Kompetitive Allele Specific PCR (KASP™). We studied 52 single nucleotide polymorphisms (SNPs) belonging to 19 genes in indigenous breeds from both countries, namely Banat's White (n = 36), Carpatina (n = 35) from Romania and Hungarian Milking (n = 79) and identified 16 polymorphic SNPs among 10 genes (PTX3, IL6, CLEC4E, IL8, IL1RN, IL15RA, TNFSF13, SOCS3, TNF and TLR3) in 150 animals. Furthermore, the diversity of the studied breeds was investigated. The PIC values ranged from 0.042 to 0.691. The mean values of observed and expected heterozygosity were 0.235 and 0.246 respectively. The highest observed heterozygosity was obtained for IL15RA g.10343904C>T in Banat's White (0.464), IL15RA g.10354813C>T in Carpatina (0.577) and SOCS3 g.52626440T>G in Hungarian Milking (0.588). Pairwise FST values between the Romanian breeds and Romanian and Hungarian breeds were small (0.009 and 0.015), indicating the close relationship among the studied goat populations. From all the polymorphic SNPs identified, the Hungarian Milking breed showed the highest proportion of polymorphisms (100%), whereas the Carpatina breed had the lowest percentage (87.5%). The highest value of MAF was obtained for SOCS3 g.52626440T>G (0.46), IL15RA g.10343904C>T (0.47), IL15RA g.10344025C>T (0.45), and IL15RA g.10354813C>T (0.42). The 16 polymorphic SNPs identified in a panel of 150 unrelated individuals belonging to three Romanian and Hungarian indigenous goat breeds could be used in future genomic based breeding schemes as markers for genetic resistance to mastitis and gastrointestinal parasitism in goat breeds found in Eastern and Central Europe.

Kompetitive Allele Specific PCR (KASP™) genotyping of 48 polymorphisms at different caprine loci in French Alpine and Saanen goat breeds and their association with milk composition.

Using a novel and fast genotyping method called Kompetitive Allele Specific PCR (KASP™), we carried out a pilot study on 48 single nucleotide polymorphisms (SNPs) belonging to 40 genes in French Alpine (n = 24) and Saanen (n = 25) goats reared in Romania. Furthermore, the associations of the 13 polymorphic genetic variants with milk production and composition were investigated. Thirty-five SNPs did not show polymorphism in the studied populations. Polymorphic SNPs were detected in the following genes: CAST, CLEC4E, DES, GHRHR, HSP90AA1, IL15RA, IL1RN, IL8, MITF, PPRC1, SOCS3, TNF and TNFSF13. The studied Alpine population was in Hardy-Weinberg disequilibrium at the g.62894878A>G locus (rs671391101) (P < 0.05). The results showed that four SNPs rs671391101 (GHRHR), rs640582069 (IL1RN) rs635583012 (SOCS3) and rs635969404 (IL15RA) out of the 13 polymorphic markers were significantly associated with milk production, protein, fat and lactose content in the Alpine breed. However, no significant effect was recorded in the Saanen population regarding milk yield or milk chemical composition. The current results provide new insights for the development of SNP marker-assisted selection technology in the goat industry and confirm the potential of using SNPs for the GHRHR, IL1RN, SOCS3, and IL15RA genes as candidate genes for selection, highlighting the direct implications of such genes for farm production outputs. The results from this study are relevant for future goat genomic studies and the inclusion of the associated traits into up-to-date selection schemes.

The influence of CSN3 and LGB polymorphisms on milk production and chemical composition in Romanian Simmental cattle.

The aim of this study was to assess the influence of genotypes from the CSN3 and LGB loci on milk production and chemical composition in Romanian Simmental cattle (n=114). For the CSN3 locus a significantly higher frequency (P≤0.001) was observed for the allele A (0.785) compared to allele B (0.215) and for AA (0.631) compared to AB (0.307) and BB (0.062) genotypes, respectively. The AA genotype was associated with a higher milk production (5887.76±115.7 kg) compared to the yields associated with the BB genotype (5619±86.34 kg, P≤0.003), as well as a higher fat percentage (4.19±0.05% vs. 4.01±0.02%, P≤0.001). A higher milk protein percentage was associated with BB (3.4±0.02%) compared to AA (3.27±0.03) and AB (3.29±0.03) genotypes, respectively (P≤0.05). For the LGB locus, the frequency of allele A was significantly higher (0.606, P≤0.008) than allele B (0.394). The AB genotype was the most prevalent in the herd (0.579) compared to AA (0.316) and BB genotypes (0.105). The AB genotype was associated with a higher milk production (5906.54±166.76 kg) compared with the other two genotypes (P≤0.05). The BB genotype was associated with a higher fat percentage in milk (4.23±0.06%) compared with AA (4.17±0.06) and AB (4.2±0.08) genotypes, respectively (P≤0.01). For the LGB locus, no significant differences (P>0.05) were observed for milk protein percentage. In order to increase the quantity and quality of milk, the outcomes obtained in this study encourage improving genetic structure in cattle based on marker assisted selection for genes with economic values.

Preliminary report on ß-casein gene Met183QVal183 polymorphism in Romanian indigenous Zackel sheep breeds.

Genetic polymorphisms of the milk protein genes are important because of their effects on quantitative traits and technological properties of milk manufacturing. In the present study we identified the polymorphism of the beta-casein gene in two local sheep breeds (Racka n=98 and Turcana n=111) in Romania. The most studied variants at the ovine beta-casein (CSN2) locus are: A and G variants. Genomic DNA was extracted from hair follicles and beta-casein genotypes were determined by the rapid TaqMan (Applied Biosystems, USA) genotyping assay. Homozygote genotypes GG were not detected in any of the studied breeds. In both, the Racka and Turcana breeds, the A variant had a much higher frequency, 0.98% and 0.97%, respectively. In the current study, the fast DNA tests for genotyping ovine CSN2 were successfully optimized, however, further samples and correlations of genomic results with milk characteristics and production data are needed for the development of future selection schemes of the Romanian indigenous sheep breeds, with the ultimate purpose to produce low allergen level sheep milk and derived dairy products.

Comparative study on production, reproduction and functional traits between Fleckvieh and Braunvieh cattle.

OBJECTIVE: Aim of the current comparative study was to evaluate production outputs, reproduction efficiency and functional traits in dual-purpose Fleckvieh and Braunvieh cows, reared under temperate European conditions. METHODS: A data-set from 414 Fleckvieh and 42 Braunvieh cows and 799 lactations was analysed. ID tag number, milk yield per milking session, number of steps/interval and milk conductivity were recorded and collected daily using AfiMilk 3.076 A-DU software (Afimilk Ltd., Kibbutz, Israel). Production and milk quality data were taken from the results of the official performance recordings and the reproductive outputs of cows were recorded by the research stations veterinarians. Comparisons between the two genotypes were carried out using the one way analysis of variance protocol, with categorical factor being considered the breed of cows. All the statistical inferences were carried out using Statistica software (StatSoft Inc., Tulsa, OK, USA). RESULTS: Fleckvieh cows significantly outperformed (p≤0.05) the Braunvieh herd, with average milk yields of 5,252.1±35.79 kg and 4,897.6±128.94 kg, respectively. Age at first calving was significantly (p≤0.01) influenced by the breed, with Fleckvieh heifers being more precocious (32.8±0.29 mo) compared to those of Braunvieh breed (35.7±0.84 mo). Reproduction efficiency as defined by the number of inseminations per gestation, calving interval, dystocia, days dry and days open, was not influenced by genotype (p>0.05). Incidences of sub-clinical mastitis, clinical mastitis, lameness and abortions were not influenced by the breed factor (p>0.05). Stayability of cows was significantly (p≤0.001) influenced by genotype, with Braunvieh cows having an average age at culling of 117.88±11.78 months compared to 90.88±2.89 months in Fleckvieh. CONCLUSION: Overall, results have shown that genotype significantly influenced milk yield, age at first calving and longevity.

Genetic polymorphism of CSN2 gene in Banat White and Carpatina goats.

The main objective of the current study was to obtain preliminary results on genetic polymorhism of A and C variants at the CSN2 locus in indigenous Romanian goat (Banat White, Carpatina) breeds using a rapid and efficient genotyping method, TaqMan assay (Applied Biosystems, USA). Hair follicle samples were taken from 73 Banat White and 82 Carpatina purebred goats from Arad, Caras-Severin and Timis counties. After the optimization of the genotyping assay it was found that the most frequent allele at the CSN2 locus was C in Banat White breed, while CSN2*A and CSN2*C showed similar frequencies in Carpatina breed (0.51 and 0.49, respectively). All three genotypes were detected in the two studied breeds, however, AA was the least frequent, especially in Banat White. The studied polymorphisms are potential markers for milk production in the studied breeds and the results will be useful in future works aimed at identifying possible associations with milk production traits, in order to test the feasibility of producing hypoallergenic organic goat milk.

Microsatellite and Mitochondrial DNA Study of Native Eastern European Cattle Populations: The Case of the Romanian Grey.

The Eastern European Grey cattle are regarded as the direct descendants of the aurochs (Bos taurus primigenius). Nowadays in Romania, less than 100 Grey animals are being reared and included in the national gene reserve. We examined the genetic diversity among Romanian Grey, Brown, Spotted and Black and White cattle breeds, with a particular focus on Romanian Grey through the use of (i) 11 bovine specific microsatellite markers on 83 animals and (ii) 638 bp length of mitochondrial DNA (mtDNA) D-loop region sequence data from a total of 81 animals. Both microsatellite and mtDNA analysis revealed a high level of genetic variation in the studied breeds. In Romanian Grey a total of 100 alleles were found, the mean number of observed alleles per locus was 9.091; the average observed heterozygosity was 0.940; the Wright's fixation index (FIS) was negative (-0.189) and indicates that there is no inbreeding and no selection pressure. MtDNA analysis revealed 52 haplotypes with 67 variable sites among the Romanian cattle breeds without any insertion or deletion. Haplotype diversity was 0.980 ± 0.007 and ranged from 0.883 ± 0.056 (Brown) to 0.990 ± 0.028 (Spotted and Black and White). The highest genetic variability of the mtDNA was recorded in the Grey breed, where 18 haplotypes were identified. The most frequent mtDNA D-loop region belonged to T3 haplogroup (80.247%), which was found across all studied breeds, while T2 haplotypes (16.049%) was only found in Grey, Spotted and Black and White genotypes. The T1 haplotypes (3.704%) were found in the Grey and Spotted. The current results contribute to the general knowledge on genetic diversity found in Eastern European cattle breeds and could prove a valuable tool for the conservation efforts of animal genetic resources (FAnGR).

Mitochondrial DNA variability in Gyimesi Racka and Turcana sheep breeds.

The current knowledge and documentation on the origins and relationship between Gyimesi Racka reared in Hungary and the Romanian Turcana is rather controversial. Lack of information and scientific reliable proofs for the divergent theories found in the two countries motivated us to implement a trial using molecular methods to assess the genetic distance and diversity in the two breeds. Hair follicles were collected from Gyimesi Racka (2 phenotypes) and from Turcana (6 ecotypes). The 599 bp segment of the D-loop region of the mitochondrial DNA was sequenced. Altogether, 42 haplotypes were identified, while 23 were found in both populations. Populations were highly diverse according to the haplotype and nucleotide diversity indices. AMOVA analysis showed that most of the variation was observed within populations (98%), indicating a weak genetic structure between the two breeds. Animals were grouped into seven groups based on their phenotype; however genetic distances among them were also low. Tajima's D, Fu's Fs, goodness-of-fit statistics, mismatch distribution and network analysis suggested recent demographic expansion. Current comprehensive mtDNA study indicates that there is very low level of genetic differentiation between the Gyimesi Racka and Turcana populations therefore they are de facto one trans-boundary breed.