BREEDING AND GENETICS SYMPOSIUM: Breeding for resilience to heat stress effects in dairy ruminants. A comprehensive review.

Selection for heat tolerant (HT) animals in dairy production has been so far linked to estimation of declines in production using milk recording and meteorological information on the day of control using reaction norms. Results from these models show that there is a reasonable amount of genetic variability in the individual response to high heat loads, which makes feasible selection of HT animals at low costs. However, the antagonistic relationship between level of production and response to heat stress (HS) implies that selection for HT animals under this approach must be done with caution so that productivity is not damaged. Decomposition of the genetic variability in principal components (PC) can provide selection criteria independent of milk production level although biological interpretation of PC is difficult. Moreover, given that response to heat stress for each animal is estimated with very sparse information collected under different physiological and management circumstances, biased (normally underestimation) and lack of accuracy may be expected. Alternative phenotypic characterization of HT can come from the use of physiological traits, which have also shown moderate heritability. However, costs of a large scale implementation based on physiological characteristics has precluded its use. Another alternative is the use of biomarkers that define heat tolerance. A review of biomarkers of HS from more recent studies is provided. Of particular interest are milk biomarkers, which together with infrared spectra prediction equations can provide useful tools for genetic selection. In the 'omics' era, genomics, transcriptomics, proteomics and metabolomics have been already used to detect genes affecting HT. A review of findings in these areas is also provided. Except for the slick hair gene, there are no other genes for which variants have been clearly associated with HT. However, integration of omics information could help in pointing at knots of the HS control network and, in the end, to a panel of markers to be used in the selection of HT animals. Overall, HT is a complex phenomenon that requires integration of fine phenotypes and omics information to provide accurate tools for selection without damaging productivity. Technological developments to make on-farm implementation feasible and with greater insight into the key biomarkers and genes involved in HT are needed.

Effect of exposure to adverse climatic conditions on production in Manchega dairy sheep.

The present study aimed to examine the effects of exposure to adverse weather conditions on milk production to assess the thermotolerance capability of the Manchega breed, a dairy sheep reared in the Mediterranean area, and the extent of decline in production outside the thermal comfort zone. To achieve this purpose, we merged data from the official milk recording of the breed with weather information and used to describe the cold and heat stress response for production traits. Production data consisted of 1,094,804 test-day records from the first 3 lactations of 177,605 ewes gathered between years 2000 to 2010. For each production trait and climate variable, the thermal load production response was characterized by the estimation of cold and heat stress thresholds that define a thermoneutral zone and the slopes of production decay outside this thermoneutral zone. Overall, we observed a comfort region between 10 and 22°C for daily average temperature, 18 and 30°C for daily maximum temperature, and from 9 to 18 units for a temperature-humidity index (THI) for all traits. Decline in production due to cold stress effects was of a greater magnitude than heat stress effects, especially for milk yield. Production losses ranged between 7 and 16 and from 0.2 to 0.6g/d per °C (or THI unit) for milk and for fat and protein yields, respectively. For heat stress, the observed decline in production was of 1 to 5 and 0.1 to 0.3g/d per °C (or THI unit) above the threshold for milk yield and for fat and protein yields, respectively. Highly productive animals showed a narrower comfort zone and higher slopes of decay. The study of lagged effects of thermal load showed how consequences of cold and heat stress are already visible in the first hours after exposure. Thus, production losses were due mainly to climate conditions on the day of control and the day before, with conditions on the previous days having a smaller effect. Annual economic losses due to thermal (cold and heat) stress ranged from 0.1 up to 4% of total profit depending on which climate variable was considered. Although of small magnitude, the effect of adverse climatic conditions on total annual farm profit is not negligible; thus, we consider the implementation of strategies aimed at reducing these losses to be important. These strategies could be target improving the mitigation strategies as well as obtaining more thermotolerant animals through selection.

Influence of semen collection method on sperm cryoresistance in small ruminants.

Semen collection for cryopreservation is a key step for small ruminant conservation programs. While in these species semen is mainly collected via artificial vagina (AV), electroejaculation (EE) provides a viable alternative for untrained males. Herein we investigated the effect of semen collection method on post-thaw sperm quality by comparing two small ruminant species, sheep and goats. Semen from Blanca-Celtibérica bucks and Manchega rams was collected by AV and EE on the same day and cryopreserved using a standard protocol. At thawing, sperm motion parameters were evaluated by CASA, whereas membrane stability (YO-PRO-1), sperm viability (propidium iodide, PI) and mitochondrial activity (Mitotracker Deep Red) were analyzed using flow cytometry. The semen collection method negatively affected post-thaw sperm quality in bucks but not in rams. Thus, in bucks, post-thaw sperm motility was higher for samples collected by AV as compared to those obtained via EE. Similarly, post-thaw sperm parameters evaluated by flow cytometry were worse for buck samples collected by EE than those collected by AV in the same species, or than ram samples regardless of collection method. These results suggest that ovine and caprine spermatozoa have a different response to the cryopreservation process depending upon the semen collection method used. We hypothesize that the EE procedure may lead to changes in the composition of the ejaculate in bucks that would make spermatozoa more susceptible to the cryopreservation process, whereas this procedure would have had no effect on ram spermatozoa. This assumption requires further investigation.

Sperm head phenotype and male fertility in ram semen.

Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter(2)/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males' fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level.

Understanding sperm heterogeneity: biological and practical implications.

Sperm are the most diverse cell type known. This diversity is thought to reflect adaptation to conditions under which sperm function as a way to ensure the survival of sperm in fertilization environments and to maximize fertilizing capacity thereof. The existence of morphological diversity among species is widely assumed, although this diversity seems less clear as we go deeper (between males, between ejaculates from the same male and even within the same ejaculate), with different theories addressing this heterogeneity. Moreover, the development of assisted reproductive techniques (ART) has led to changes in the physiological conditions in which sperm fertilize, which could lead, ultimately, to a selection towards more favourable sperm design. Regardless of the origin of this diversity, when studying the relationship between shape and function of sperm, it is advisable to assess the degree of heterogeneity of sperm and takes into account to be more likely to identify those morphological characteristics determining the fertile ability of sperm. Otherwise, these relationships could be hidden as a result of considering an average shape not representative of morphological characteristics of sperm. In addition, the knowledge of this morphological diversity in terms of changes arising from modifications in the sperm environment and mechanisms that generate these changes could be useful for understanding the reproductive capacity of males but also in enhancing their fertile ability.

Genomewide association for a dominant pigmentation gene in sheep.

Most published genomewide association studies (GWAS) in sheep have investigated recessively inherited monogenic traits. The objective here was to assess the feasibility of performing GWAS for a dominant trait for which the genetic basis was already known. A total of 42 Manchega and Rasa Aragonesa sheep that segregate solid black or white coat pigmentation were genotyped using the SNP50 BeadChip. Previous analysis in Manchegas demonstrated a complete association between the pigmentation trait and alleles of the MC1R gene, setting an a priori expectation for GWAS. Multiple methods were used to identify and quantify the strength of population substructure between black and white animals, before allelic association testing was performed for 49,034 SNPs. Following correction for substructure, GWAS identified the most strongly associated SNP (s26449) was also the closest to the MC1R gene. The finding was strongly supported by the permutation tree-based random forest (RF) analysis. Importantly, GWAS identified unlinked SNP with only slightly lower p-values than for s26449. Random forest analysis indicated these were false positives, suggesting interpretation based on both approaches was beneficial. The results indicate that a combined analytical approach can be successful in studies where a modest number of animals are available and substantial population stratification exists.

Single layer centrifugation (SLC) improves sperm quality of cryopreserved Blanca-Celtibérica buck semen.

The aim of the present study was to evaluate the effect of sperm selection by means of single layer centrifugation (SLC) on sperm quality after cryopreservation, either when SLC is used before freezing or after thawing, using Blanca-Celtibérica buck semen collected by electroejaculation (EE). Ejaculates from six bucks were collected by EE and divided into two aliquots. One of them (unselected) was diluted with Biladyl(®) by the two-step method and frozen over nitrogen vapor. The other aliquot was selected by the SLC technique and subsequently frozen in the same way as the unselected samples (SLC before freezing). In a further treatment, two unselected straws were thawed and SLC was carried out (SLC after thawing). At thawing, sperm motility of all samples ((i) unselected; (ii) selected before freezing and (iii) selected after thawing) was evaluated by CASA. In addition, integrity of the plasma membrane, mitochondrial membrane potential, ROS production and DNA fragmentation index were assessed by flow cytometry. Most of the sperm parameters were improved (P≤0.001) in samples selected by SLC after thawing in relation to unselected or selected by SLC before freezing. The percentage of progressive motile spermatozoa was greater (86%) for sperm samples selected after thawing compared with unselected (58%) or selected before freezing (54%). Moreover, percentages of spermatozoa with intact plasma membrane and spermatozoa with high mitochondrial membrane potential (hMMP) were also greater for sperm samples selected after thawing compared to sperm samples unselected or selected before freezing (spermatozoa with intact plasma membrane: 80% vs. 32% vs. 12%; spermatozoa with hMMP: 54% vs. 1% vs. 15%; respectively). Therefore, sperm quality after cryopreservation is improved in Blanca-Celtibérica buck ejaculates collected by EE when a sperm selection technique such as SLC is carried out after thawing.

Effect of semen collection method (artificial vagina vs. electroejaculation), extender and centrifugation on post-thaw sperm quality of Blanca-Celtibérica buck ejaculates.

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl(®), Andromed(®) and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1-/PI- (intact spermatozoa), and Mitotracker+/YO-PRO-1- (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl(®) and Andromed(®) resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl(®) or Andromed(®) are used as freezing extenders.

Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in ram ejaculates: intra- and interindividual variation.

The existence of sperm subpopulations within the mammalian ejaculate has now been widely recognized. However, to the best of our knowledge, no data exist regarding the existence of sperm morphometric subpopulations within the ovine ejaculate. Computer assisted sperm morphometry analysis (ASMA) data and clustering methods were used in this study to identify sperm-head subpopulations in ram semen. Two experiments were carried out. In Experiment 1, ejaculates from 226 mature rams of the Manchega breed belonging to 36 different herds were used. A minimum of 100 sperm heads were analyzed from each male and eight morphometric characteristics for each individual sperm were recorded. Subpopulation analysis was performed in sequential steps: variable group analysis and correlation analysis to select which morphometric characteristics to use in cluster analyses; nonhierarchical clustering analysis using sperm head length and p2a (also known as roundness) shape factor as initial classificatory variables; and hierarchical clustering analysis to obtain the final number of clusters. The clustering analyses, based on 26,306 individual cells, revealed the existence of four sperm subpopulations (SP1, SP2, SP3 and SP4) with different morphometric characteristics. Significant differences in the proportion of spermatozoa in the SP1 and SP3 were found between rams belonging to different herds. In Experiment 2, the intra- and intermale variability on the distribution of sperm subpopulations was assessed. Three ejaculates from each of 21 rams were collected and the same multistep clustering analysis was performed. For all subpopulations defined, the intermale variability resulted in high values, being the intramale variability much lower. This fact would allow the use of sperm head morphometry to characterize a male and might provide valuable information to asses its fertility. In conclusion, our results show that using computer assisted sperm morphometry analysis and multivariate cluster analyses, four sperm subpopulations with different head phenotype were identified in ram ejaculates.

Effect of storage temperature during transport of ovaries on in vitro embryo production in Iberian red deer (Cervus elaphus hispanicus).

The aim of this work was to study the effect of storage temperature during the transport of ovaries on cleavage and blastocyst rates in Iberian red deer, because wild populations of this subspecies are usually far from laboratories. A total of 472 ovaries from 236 Iberian hinds were recovered and maintained in saline solution at 5-8 °C or 20-25 °C for 12 h. After storage, aspirated oocytes were matured with FSH/LH or EGF and the developed embryos were cultured with oviduct epithelial cells monolayer (OCM). A higher (P = 0.009) cleavage rate was obtained when the ovaries were stored at 5-8 °C. However, there were no differences between both storage temperatures in relation to the percentage of blastocysts obtained. Considering the management and production systems of Iberian red deer, this study provides important information about the ovary storage temperature during transport with the purpose of assuring an optimal in vitro embryo production.

Economic weights for major milk constituents of Manchega dairy ewes.

The objective of this study was to estimate economic weights of major components of milk (fat and protein) for the Manchega dairy sheep breed. An economic study was carried out and the profit associated with fat and protein yields of milk was calculated as the difference between incomes and costs. Incomes were obtained from milk sales to cheese industry and a reference marked price was used. Costs were calculated considering the energy necessary to produce each of the components of milk, and the price per milk forage unit was calculated as the total expense in feedstuff divided by the total (theoretical) needs of the flock. Economic values were defined as partial derivatives of the profit function with respect to each trait. Economic weights for fat and protein yields were similar, being slightly greater for protein in all cases. For carrier, economic weights were close to zero and negative because an increase in carrier production without changes in fat and protein composition leads to an increase in energy demands, holding the sale price of milk constant. When genetic standard deviations were taken into account and standardized economic values were calculated, an increase in economic value of protein and a decrease in economic value of fat yields were observed. The consequences that different changes in production system conditions have on the estimated economic weights were also studied. In general, economic weights were relatively insensitive to changes in production levels and market prices except for changes on milk price. Given the economic importance shown for fat and protein, milk components should be taken into consideration when breeding objectives for dairy sheep are established.

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa.

The aim of the present study was to evaluate the effect of selecting a sperm subpopulation by means of a discontinuous density gradient centrifugation (DGC) on the quality of ram thawed semen, and the relationships between sperm parameters assessed in unselected and in selected sperm samples with in vivo fertility after intrauterine artificial insemination (IUI) using unselected sperm samples. Semen samples from twenty males were collected by artificial vagina and cryopreserved following a standard protocol. After thawing, unselected sperm samples were used in an in vivo fertility trial and sperm motility (subjective and objective, assessed by means of CASA) and membrane and acrosomal integrities (microscopy) were evaluated on unselected and selected sperm samples. In addition, plasmalemma integrity (YO-PRO-1/PI), membrane fluidity (Merocyanine 540/YO-PRO-1), mitochondrial activity (Mitotracker Deep Red/YO-PRO-1), and DNA fragmentation index (%DFI) assessed by Sperm Chromatin Structure Assay (SCSA) were evaluated by flow cytometry before and after sperm processing using DGC. Results showed that DGC improved all sperm parameters significantly, except the %DFI, which increased after the selection procedure. No relationships were found between sperm parameters evaluated in unselected sperm samples and in vivo fertility. However, we found a positive correlation between spermatozoa with high membrane fluidity within the viable sperm population (VIABMerocyanine+) evaluated in selected sperm samples and in vivo fertility (r = 0.370, P = 0.019). In conclusion, our results suggest that selected spermatozoa represent a sperm subpopulation different to the unselected one that could be related with the in vivo fertility.

Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer.

Sperm morphology has been identified as a characteristic that can be useful in the prediction of fertilizing capacity. The aim of the current study was to characterize ram sperm heads morphometrically as a basis for future studies on the relationship between sperm quality and male fertility. For this purpose, ejaculates from 241 mature rams (Ovis aries) belonging to 36 different dairy herds were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer. Sperm samples, collected by artificial vagina, were diluted in phosphate buffered saline (PBS) for the analysis. A microscope slide was prepared from single-diluted fresh sperm samples. Slides were air-dried and stained with Hemacolor. A minimum of 115 sperm heads were analyzed from each male. Each sperm head was measured for four primary parameters (area, perimeter, length, width), and four derived parameters of head shape were obtained. Significant differences in sperm head morphometry were found between rams (CV for morphometric parameters ranging from 0.9 to 10.1), and there were marked differences in the sperm morphometric composition of the ejaculates. For all parameters, within-animal CVs were greater than between-animal CVs. Within-animal CVs ranged from 4.2 to 10.6, showing the high degree of sperm polymorphism present in the sheep ejaculate. Significant differences in sperm head morphometry were found between rams belonging to the different herds (i.e., origin). An important part of the variability observed on morphometric parameters was due to the male itself, with an explained variance ranging from 3.6% for regularity to 34.0% for p2a (perimeter(2)/[4xpixarea]). The explained variance by the herd of origin of the males ranged from 0.6% for regularity to 10.8% for area. Our results suggest that a genetic component might be responsible for the observed sperm head morphometry differences between herds.

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: electroejaculation and postmortem collection.

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1-/PI-) was higher for postmortem samples (P<0.001), although the ratio of YO-PRO-1- spermatozoa within the PI- population was higher for ejaculated samples (P=0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA-) was higher for postmortem samples (P<0.001 and P<0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA- cells was higher for electroejaculated samples (P=0.026 and P=0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P=0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.

Heterologous in vitro fertilization is a good procedure to assess the fertility of thawed ram spermatozoa.

A heterologous in vitro fertilization (IVF) test using calf oocytes with zona pellucida was employed to assess the fertility of thawed ram sperm samples. Six males with significant differences in fertility (P=0.003) were used. The males were classified as having high fertility (>or=42%) and low fertility (

Economic weights of fertility, prolificacy, milk yield and longevity in dairy sheep.

Economic weights have been estimated in two breeds (Latxa and Manchega) using economic and technical data collected in 41 Latxa and 12 Manchega dairy sheep flocks. The traits considered were fertility (lambing per year), prolificacy (number of lambs), milk yield (litres) and longevity (as productive life, in years). A linear function was used, relating these traits to the different costs in the flock. The variable costs involved in the profit function were feed and labour. From this function, economic weights were obtained. Labour is considered in the Latxa breed to be a constraint. Moreover, farm profits are unusually high, which probably means that some costs were not included according to the economic theory. For that reason, a rescaling procedure was applied constraining total labour time at the farm. Genetic gains were estimated with the resulting economic weights to test if they give any practical difference. Milk yield only as selection criterion was also considered. The medians of the estimated economic weights for fertility, prolificacy, milk yield and longevity were 138.60 € per lambing, 40.00 € per lamb, 1.18 € per l, 1.66 € per year, and 137.66 € per lambing, 34.17 € per lamb, 0.73 € per l, 2.16 € per year under the linear approach in the Latxa and Manchega breeds respectively. Most differences between breeds can be related to differences in production systems. As for the genetic gains, they were very similar for all economic weights, except when only milk yield was considered, where a correlated decrease in fertility led to a strong decrease in profit. It is concluded that the estimates are robust for practical purposes and that breeding programmes should consider inclusion of fertility. More research is needed to include other traits such as somatic cell score, milk composition and udder traits.

Economic weights of somatic cell score in dairy sheep.

The economic weights for somatic cell score (SCS) have been calculated using profit functions. Economic data were collected in the Latxa breed. Three aspects have been considered: bulk tank milk payment, veterinary treatments due to high SCS, and culling. All of them are non-linear profit functions. Milk payment is based on the sum of the log-normal distributions of somatic cell count, and veterinary treatments on the probability of subclinical mastitis, which is inferred when individual SCS surpass some threshold. Both functions lead to non-standard distributions. The derivatives of the profit function were computed numerically. Culling was computed by assuming that a conceptual trait culled by mastitis (CBM) is genetically correlated to SCS. The economic weight considers the increase in the breeding value of CBM correlated to an increase in the breeding value of SCS, assuming genetic correlations ranging from 0 to 0.9. The relevance of the economic weights for selection purposes was checked by the estimation of genetic gains for milk yield and SCS under several scenarios of genetic parameters and economic weights. The overall economic weights for SCS range from - 2.6 to - 9.5 € per point of SCS, with an average of - 4 € per point of SCS, depending on the expected average SCS of the flock. The economic weight is higher around the thresholds for payment policies. Economic weights did not change greatly with other assumptions. The estimated genetic gains with economic weights of 0.83 € per l of milk yield and - 4 € per point of SCS, assuming a genetic correlation of - 0.30, were 3.85 l and - 0.031 SCS per year, with an associated increase in profit of 3.32 €. This represents a very small increase in profit (about 1%) relative to selecting only for milk yield. Other situations (increased economic weights, different genetic correlations) produced similar genetic gains and changes in profit. A desired-gains index reduced the increase in profit by 3%, although it could be greater depending on the genetic parameters. It is concluded that the inclusion of SCS in dairy sheep breeding programs is of low economic relevance and recommended only if recording is inexpensive or for animal welfare concerns.