Analysis of Varroa Mite Colony Infestation Level Using New Open Software Based on Deep Learning Techniques.

Varroa mites, scientifically identified as Varroa destructor, pose a significant threat to beekeeping and cause one of the most destructive diseases affecting honey bee populations. These parasites attach to bees, feeding on their fat tissue, weakening their immune systems, reducing their lifespans, and even causing colony collapse. They also feed during the pre-imaginal stages of the honey bee in brood cells. Given the critical role of honey bees in pollination and the global food supply, controlling Varroa mites is imperative. One of the most common methods used to evaluate the level of Varroa mite infestation in a bee colony is to count all the mites that fall onto sticky boards placed at the bottom of a colony. However, this is usually a manual process that takes a considerable amount of time. This work proposes a deep learning approach for locating and counting Varroa mites using images of the sticky boards taken by smartphone cameras. To this end, a new realistic dataset has been built: it includes images containing numerous artifacts and blurred parts, which makes the task challenging. After testing various architectures (mainly based on two-stage detectors with feature pyramid networks), combination of hyperparameters and some image enhancement techniques, we have obtained a system that achieves a mean average precision (mAP) metric of 0.9073 on the validation set.

Bull Sperm SWATH-MS-Based Proteomics Reveals Link between High Fertility and Energy Production, Motility Structures, and Sperm-Oocyte Interaction.

The prediction of male or semen fertility potential remains a persistent challenge that has yet to be fully resolved. This work analyzed several in vitro parameters and proteome of spermatozoa in bulls cataloged as high- (HF; n = 5) and low-field (LF; n = 5) fertility after more than a thousand artificial inseminations. Sperm motility was evaluated by computer-assisted sperm analysis. Sperm viability, mitochondrial membrane potential (MMP) and reactive oxygen species (mROS) of spermatozoa were assessed by flow cytometry. Proteome was evaluated by the SWATH-MS procedure. Spermatozoa of HF bulls showed significantly higher total motility than the LF group (41.4% vs 29.7%). Rates of healthy sperm (live, high MMP, and low mROS) for HF and LF bull groups were 49% and 43%, respectively (p > 0.05). Spermatozoa of HF bulls showed a higher presence of differentially abundant proteins (DAPs) related to both energy production (COX7C), mainly the OXPHOS pathway, and the development of structures linked with the motility process (TPPP2, SSMEM1, and SPAG16). Furthermore, we observed that equatorin (EQTN), together with other DAPs related to the interaction with the oocyte, was overrepresented in HF bull spermatozoa. The biological processes related to protein processing, catabolism, and protein folding were found to be overrepresented in LF bull sperm in which the HSP90AA1 chaperone was identified as the most DAP. Data are available via ProteomeXchange with identifier PXD042286.

OpenCASA: A new open-source and scalable tool for sperm quality analysis.

In the field of assisted reproductive techniques (ART), computer-assisted sperm analysis (CASA) systems have proved their utility and potential for assessing sperm quality, improving the prediction of the fertility potential of a seminal dose. Although most laboratories and scientific centers use commercial systems, in the recent years certain free and open-source alternatives have emerged that can reduce the costs that research groups have to face. However, these open-source alternatives cannot analyze sperm kinetic responses to different stimuli, such as chemotaxis, thermotaxis or rheotaxis. In addition, the programs released to date have not usually been designed to encourage the scalability and the continuity of software development. We have developed an open-source CASA software, called OpenCASA, which allows users to study three classical sperm quality parameters: motility, morphometry and membrane integrity (viability) and offers the possibility of analyzing the guided movement response of spermatozoa to different stimuli (useful for chemotaxis, thermotaxis or rheotaxis studies) or different motile cells such as bacteria, using a single software. This software has been released in a Version Control System at Github. This platform will allow researchers not only to download the software but also to be involved in and contribute to further developments. Additionally, a Google group has been created to allow the research community to interact and discuss OpenCASA. For validation of the OpenCASA software, we analysed different simulated sperm populations (for chemotaxis module) and evaluated 36 ejaculates obtained from 12 fertile rams using other sperm analysis systems (for motility, membrane integrity and morphology modules). The results were compared with those obtained by Open-CASA using the Pearson's correlation and Bland-Altman tests, obtaining a high level of correlation in all parameters and a good agreement between the different used methods and the OpenCASA. With this work, we propose an open-source project oriented to the development of a new software application for sperm quality analysis. This proposed software will use a minimally centralized infrastructure to allow the continued development of its modules by the research community.

Microbiota Analysis of Ejaculated Honey Bee Drone Semen and the Effect of Semen Collection Method on Bacterial Loads.

Artificial insemination in queen honey bees is the only tool that provides complete control over mating for research and breeding purposes, making it essential in genetic improvement and conservation programs in this species. The aims of this study were to characterize drone semen bacterial loads by culture-dependent and independent methods and to describe their variation depending on the method of semen collection, the colony and the apiary. In the first experiment, the bacterial loads of semen collected from the seminal vesicles or from ejaculates was studied using culture-dependent methods. The collection method had a significant influence on the overall bacterial count in semen. Out of the 42 semen samples analyzed, 26 (61.9%) tested positive for bacterial isolation. This encompassed the entirety of samples obtained from the seminal vesicles (21 of 21), whereas only 23.8% of those derived from ejaculates (5 out of 21) showed bacterial isolation. In the second experiment, next-generation sequencing techniques were used to describe the microbiome of ejaculated drone semen for the first time. The most abundant phyla were Proteobacteria, Firmicutes, Bacteroidota and Actinobacteriota, while the most abundant genera were Lactobacillus, Staphylococcus, Prevotella, Alloprevotella and Streptococcus. The results showed that the apiary had a significant effect on the community structure composition and abundance of the seminal microbiota, and significative differences in abundance were observed for the genera Sphingomonas, Methylobacterium-Methylorubrum, Bifidobacterium and Alloprevotella. Significant differences were also observed in the richness of the microbiota between apiaries and colonies.

Understanding Sperm Quality for Improved Reproductive Performance.

The assessment of semen quality is used to identify factors that influence sperm performance and diagnose male infertility [...].

Relationships between body weight and milk yield during the early postpartum period and bull and technician and the reproductive performance of high producing dairy cows.

The aim of this study was to evaluate the effect of body weight (BW) change during the early postpartum period and BW on day 50 post partum on the subsequent expression of estrus and fertility at first insemination before day 90 post partum. The data were derived from 1036 postpartum cows reaching 90 days in milk. The following data were recorded for each animal: lactation number, daily milk production at day 50 post partum, calving date, days in milk at estrus and at AI, insemination date, insemination number, inseminating bull, AI technician and body weight at calving, at day 50 post partum and at AI. Of the 1036 cows registered, 817 (78.9%) showed first estrus before day 90 post partum and were inseminated. Using logistic regression procedures and based on the odds ratio a one unit (kg) increase in the daily milk production at day 50 post partum and a one unit (kg) decrease in the BW between calving and day 50 post partum were related to a 1.03-fold increase and a 0.97-fold decrease in the estrus expression rate before day 90 post partum, respectively. Of the 817 cows inseminated before day 90 post partum, 437 became pregnant (53.5%). A one unit increase in the BW at day 50 post partum produced a 1.003-fold increase in the early fertility rate. Pregnancy rate before day 90 was also influenced by the season at calving, bull providing semen and technician. In conclusion, the register of BW during the postpartum period may be a useful tool for evaluating the nutritional status and its relationship with the subsequent reproductive efficiency in dairy cows.

Consumers' willingness to pay for beef direct sales. A regional comparison across the Pyrenees.

Willingness to pay (WTP) for direct market of beef is investigated in two Spanish and two French regions located on both sides of the Pyrenees. Given the novelty of this distribution system, especially in Spain, a contingent valuation approach is undertaken, and a double-bounded model is estimated. Different patterns of awareness, use and WTP are found across regions. Likewise, the profile of current and potential users of direct sale chains is investigated. Experience in the different stages involved from choice to final consumption of beef, intensity of varied beef consumption, familiarity with direct market of food in general, and beef in particular, are some of the relevant factors to explain WTP and the probability of getting engaged into a direct distribution system of beef.

Effect of the Intake of Lean Red-Meat from Beef-(Pirenaica Breed) versus Lean White-Meat on Body Composition, Fatty Acids Profile and Cardiovascular Risk Indicators: A Randomized Cross-Over Study in Healthy Young Adults.

The main dietary guidelines recommended a restriction of total and saturated fat intake in the management of cardiovascular risk. These recommendations are overgeneralized, and all red meats should be limited and replaced by white meat. The aim is to assess the effect of the consumption of beef (from the Pirenaica breed) or chicken-based diets on body composition, fatty acid profile and cardiovascular (CV) risk indicators in healthy adults. A randomized cross-over study was carried out in three University accommodation halls. Participants consumed either the Pirenaica breed beef or chicken three times per week for 8-week periods with their usual diet. Body composition, clinical, biochemical and dietary variables were evaluated at baseline and at the end of each period. A validated diet questionnaire was used to assess nutrient intake and monitor compliance. Intervention and control group comparisons were done with the general linear regression model for repeated measures. Forty-seven healthy adults were included (51.6% males, mean age 19.9 ± 1.75 years). No significant differences were found in body composition, fatty acid profile or CV risk indicators from baseline in either diet group. Consumption of lean red meat (Pirenaica breed) or lean white meat (chicken) as part of the usual diet is associated with a similar response. Clinical Trial Registration: NCT04832217 (accessed on 6 September 2022).

Role of Antioxidants in Cooled Liquid Storage of Mammal Spermatozoa.

Cooled preservation of semen is usually associated with artificial insemination and genetic improvement programs in livestock species. Several studies have reported an increase in reactive oxidative species and a decrease in antioxidant substances and sperm quality parameters during long-term semen storage at refrigerated temperatures. The supplementation of antioxidants in extenders before refrigeration could reduce this detrimental effect. Various antioxidants have been tested, both enzymatic, such as superoxide dismutase and catalase, and non-enzymatic, such as reduced glutathione, vitamins E and C and melatonin. However, the problem of oxidative stress in semen storage has not been fully resolved. The effects of antioxidants for semen-cooled storage have not been reviewed in depth. Therefore, the objective of the present study was to review the efficiency of the supplementation of antioxidants in the extender during cooled storage of semen in livestock species.

Ability of the ISAS3Fun Method to Detect Sperm Acrosome Integrity and Its Potential to Discriminate between High and Low Field Fertility Bulls.

The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls were tested; 10 bulls had high field fertility and 10 bulls had low field fertility. Analyses of sperm motility, membrane integrity, and membrane-acrosome integrity with the ISAS3Fun method were performed. Sperm morphometry of the fluorescence sperm subpopulations obtained with the ISAS3Fun method was also analysed. Significant differences between high- and low-fertility groups were only found with the ISAS3Fun technique, specifically in sperm acrosome integrity, the proportion of spermatozoa with an intact acrosome and damaged membrane, and in sperm head width of spermatozoa with intact structures. Discriminant analyses allowed us to correctly classify 90% of sperm samples in their fertility group using sperm quality parameters. Given that only the results obtained with the ISAS3Fun technique were related to bull fertility, we performed a second experiment aimed to validate the efficacy of this technique to detect the acrosomal integrity of bull spermatozoa, comparing them with the conventional FITC-PNA/propidium iodide (PNA/PI) combination under capacitating conditions. The results indicated that the ISAS3Fun combination provided an accurate assessment of both viability and acrosomal integrity for ejaculated spermatozoa, while the PNA/PI combination underestimated the extension of acrosomal damage due to false negatives. It was concluded that the simultaneous assessment of sperm plasma membranes and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than more conventional in vitro sperm quality tests.

Revisiting Cattle Temperament in Beef Cow-Calf Systems: Insights from Farmers' Perceptions about an Autochthonous Breed.

Understanding temperament is an important part of cattle production since undesirable temperament may cause serious problems associated with aggression, maternal care, and human safety. However, little is known about how farmers define or assess temperament, especially in autochthonous cattle breeds. The aim of this study was to explore perceptions of farmers about the temperament of the Pyrenean cattle breed with special attention to beef cow-calf systems in Spain. The methodology used to obtain the information was focus group discussions (FGD). Farmers defined temperament as a behavioural response to challenging situations imposed by human handling. Specific terms used were related to active or passive reactions to fear (e.g., "strong", "aggressive", "nervous", "fearful"). The speed of response to stimuli was also important. Female temperament was thought to become more docile with age while bull temperament was more variable. Maternal aggressiveness was highlighted as a potential human safety problem, but also desirable in an extensively bred animal who may need to defend calves against predators. Anatomical characteristics were seen as unreliable predictors of temperament, while behavioural indicators were more widely used, such as "alertness", which was a general trait of the breed, and "gaze", which, when associated with an alert expression, suggests a potential threat. Sensory acuity, such as sight and smell, were thought to be related with temperament in some FGDs but there was no overall agreement as to whether different behavioural responses were due to differences in sensory acuity. The results from the study could be useful during training programs or in the development of new genetic selection schemes and evaluation protocols involving cattle temperament.

Effects of cumulative stressful and acute variation episodes of farm climate conditions on late embryo/early fetal loss in high producing dairy cows.

The aim of this study was to determine possible relationships between farm climate conditions, recorded from day 0 to day 40 post-artificial insemination (AI), and late embryo/early fetal loss in high producing dairy cows. Pregnancy was diagnosed by rectal ultrasonography between 28 and 34 days post-AI. Fetal loss was registered when a further 80- to 86-day diagnosis proved negative. Climate variables such as air temperature and relative humidity (RH) were monitored in the cubicles area for each 30-min period. Temperature-humidity indices (THI); cumulative stressful values and episodes of acute change (defined as the mean daily value 1.2 times higher or lower than the mean daily values of the 10 previous days) of the climate variables were calculated. The data were derived from 759 cows in one herd. A total of 692 pregnancies (91.2%) carried singletons and 67 (8.8%) carried twins. No triplets were recorded. Pregnancy loss was recorded in 6.7% (51/759) of pregnancies: 5.6% (39/692) in single and 17.9% (12/67) in twin pregnancies. Using logistic regression procedures, a one-unit increase in the daily cumulative number of hours for the THI values higher than 85 during days 11-20 of gestation caused a 1.57-fold increase in the pregnancy loss, whereas the likelihood of fetal loss increased by a factor of 1.16 for each additional episode of acute variation for the maximum THI values during gestation days 0-40. THI values higher than 85 and episodes of acute variation for the maximum THI values were only recorded during the warm and cool periods, respectively. The presence of twins led to a 3.98-fold increase in pregnancy loss. In conclusion, our findings show that cumulative stressful and episodes of acute variation of climatic conditions can compromise the success of gestation during both the cool and warm periods of the year. Twin pregnancy was confirmed as a main factor associated with pregnancy loss.

Sperm Quality Assessment in Honey Bee Drones.

The quality of honey bee drone semen is relevant in different contexts, ranging from colony productivity to pathology, toxicology and biodiversity preservation. Despite its importance, considerably less knowledge is available on this subject for the honey bee when compared to other domestic animal species. A proper assessment of sperm quality requires a multiple testing approach which discriminates between the different aspects of sperm integrity and functionality. Most studies on drone semen quality have only assessed a few parameters, such as sperm volume, sperm concentration and/or sperm plasma membrane integrity. Although more recent studies have focused on a broader variety of aspects of semen quality, some techniques currently used in vertebrates, such as computer-assisted sperm analysis (CASA) or multiparametric sperm quality testing, still remain to be developed in the honey bee. This may be attributed to the particular sperm morphology and physiology in this species, requiring the development of technologies specifically adapted to it. This article reviews the present knowledge of sperm quality in honey bee drones, highlighting its peculiarities and proposing future lines of research.

Relationship of sperm plasma membrane and acrosomal integrities with sperm morphometry in Bos taurus.

To date, sperm morphometric studies have assessed whole sperm populations without considering sperm function. The aim of this study was to evaluate the relationship of sperm membrane and acrosomal integrity with sperm morphometry in liquid semen samples collected from bulls. To this end, sperm morphometry was performed on cryopreserved semen samples from 16 bulls by a combination of fluorescent dyes, including Hoechst 33343, carboxyfluorescein diacetate, and propidium iodide. This allowed discrimination of different subpopulations on the basis of sperm membrane and acrosomal integrity and analysis of the morphometrics of the sperm head, nucleus, and acrosome using a specific plug-in module created on ImageJ. Acrosomal integrity was related to sperm morphometry as the heads of spermatozoa with a damaged acrosome were significantly smaller than those with a normal acrosome (P < 0.001). In the case of spermatozoa with an intact acrosome, those with a damaged plasma membrane had a larger sperm head and acrosome than spermatozoa with an intact plasma membrane (P < 0.001). No significant differences in the sperm head size were observed between sperm subpopulations without an acrosome or in the nuclear sperm morphometry of the different subpopulations. There was a positive correlation between the sperm motility values of the samples and the morphometric parameters for intact spermatozoa. These correlations were particularly strong for the morphometric parameters of the sperm acrosome. We conclude that there are clear differences in the sperm morphometry depending on the status of the sperm membrane and acrosome and this should be considered when performing this kind of analysis.

Low seroprevalence of Neospora caninum infection associated with the limousin breed in cow-calf herds in Andorra, Europe.

Neospora caninum seroprevalence and risk factors affecting seroprevalence in beef cattle in Andorra were investigated. Antibodies to N. caninum were evaluated by enzyme-linked immunosorbent assay performed on a yearly basis in 1,758 animals older than 6 mo, belonging to 26 herds. Mean seroprevalence of antibodies to N. caninum for the herds was 7.4 +/- 1.2% (130/1,758). Logistic regression analyses were performed on data from each animal, considering N. caninum seropositivity as the dependent variable, and herd, grazing area, year of sampling, repeat-test animal (animals sampled twice or more), sex, breed, age (animals <4 yr old or > or =5 yr old), and country of birth as possible risk factors. Based on the odds ratio, the prevalence of infection was 2.1 times higher (P < 0.01) in animals from the Ordino grazing area, 1.64 times higher in animals older than 5 yr (P < 0.01), and 6.7 times (1/0.15) lower in Limousin-mixed Limousin cattle (P < 0.002). The results suggest that the particular grazing location could promote the horizontal transmission of this parasite and that certain breeds are less susceptible to N. caninum infection than others.

Morphometric and kinematic sperm subpopulations in split ejaculates of normozoospermic men.

This study was designed to analyze the sperm kinematic and morphometric subpopulations in the different fractions of the ejaculate in normozoospermic men. Ejaculates from eight normozoospermic men were collected by masturbation in three fractions after 3-5 days of sexual abstinence. Analyses of sperm motility by computer-assisted sperm analysis (CASA-Mot), and of sperm morphometry by computer-assisted sperm morphometry analysis (CASA-Morph) using fluorescence were performed. Clustering and discriminant procedures were performed to identify sperm subpopulations in the kinematic and morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three kinematic subpopulations (slow with low ALH [35.6% of all motile spermatozoa], with circular trajectories [32.0%], and rapid with high ALH [32.4%]), and three morphometric subpopulations (large-round [33.9% of all spermatozoa], elongated [32.0%], and small [34.10%]). The distribution of kinematic sperm subpopulations was different among ejaculate fractions (P < 0.001), with higher percentages of spermatozoa exhibiting slow movements with low ALH in the second and third portions, and with a more homogeneous distribution of kinematic sperm subpopulations in the first portion. The distribution of morphometric sperm subpopulations was also different among ejaculate fractions (P < 0.001), with more elongated spermatozoa in the first, and of small spermatozoa in the third, portion. It is concluded that important variations in the distribution of kinematic and morphometric sperm subpopulations exist between ejaculate fractions, with possible functional implications.

A comparative study of the morphometry of sperm head components in cattle, sheep, and pigs with a computer-assisted fluorescence method.

The aim of this study was to compare the sperm nuclear and acrosomal morphometry of three species of domestic artiodactyls; cattle (Bos taurus), sheep (Ovis aries), and pigs (Sus scrofa). Semen smears of twenty ejaculates from each species were fixed and labeled with a propidium iodide-Pisum sativum agglutinin (PI/PSA) combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed. The use of the PI/PSA combination and CASA-Morph fluorescence-based method allowed the capture, morphometric analysis, and differentiation of most sperm nuclei, acrosomes and whole heads, and the assessment of acrosomal integrity with a high precision in the three species studied. For the size of the head and nuclear area, the relationship between the three species may be summarized as bull > ram > boar. However, for the other morphometric parameters (length, width, and perimeter), there were differences in the relationships between species for sperm nuclei and whole sperm heads. Bull sperm acrosomes were clearly smaller than those in the other species studied and covered a smaller proportion of the sperm head. The acrosomal morphology, small in the bull, large and broad in the sheep, and large, long, and with a pronounced equatorial segment curve in the boar, was species-characteristic. It was concluded that there are clear variations in the size and shape of the sperm head components between the three species studied, the acrosome being the structure showing the most variability, allowing a clear distinction of the spermatozoa of each species.

Comparison of different statistical approaches to evaluate morphometric sperm subpopulations in men.

This study was designed to characterize morphometric sperm subpopulations in normozoospermic men by using different statistical methods and examining their suitability to classify correctly different sperm nuclear morphologies present in human ejaculates. Ejaculates from 21 normozoospermic men were collected for the study. After semen collection and analysis, samples were prepared for morphometric determination. At least 200 spermatozoa per sample were assessed for sperm morphometry by computer-assisted sperm morphometry analysis (CASA-Morph) using fluorescence. Clustering and discriminant procedures were performed to identify sperm subpopulations from the morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three morphometric subpopulations (large-round 30.4%, small-round 46.6%, and large-elongated 22.9%). In the second analysis, using discriminant methods, the classification was made independently of size and shape. Three morphological categories according to nuclear size (small <10.90 µm2, intermediate 10.91-13.07 µm2, and large >13.07 µm2) and four categories were defined on 400 canonical cells (100 × 4) from 10 men according to sperm nuclear shape (oval, pyriform, round, and elongated). Thereafter, the resulting classification functions were used to categorize 4200 spermatozoa from 21 men. Differences in the class distribution were observed among men from both clustering and discriminant procedures. It was concluded that the combination of CASA-Morph fluorescence-based technology with multivariate cluster or discriminant analyses provides new information on the description of different morphometric sperm subpopulations in normal individuals, and that important variations in the distribution of morphometric sperm subpopulations may exist between men, with possible functional implications.

Computer-assisted sperm morphometry fluorescence-based analysis has potential to determine progeny sex.

This study was designed to determine the ability of computer-assisted sperm morphometry analysis (CASA-Morph) with fluorescence to discriminate between spermatozoa carrying different sex chromosomes from the nuclear morphometrics generated and different statistical procedures in the bovine species. The study was divided into two experiments. The first was to study the morphometric differences between X- and Y-chromosome-bearing spermatozoa (SX and SY, respectively). Spermatozoa from eight bulls were processed to assess simultaneously the sex chromosome by FISH and sperm morphometry by fluorescence-based CASA-Morph. SX cells were larger than SY cells on average (P < 0.001) although with important differences between bulls. A simultaneous evaluation of all the measured features by discriminant analysis revealed that nuclear area and average fluorescence intensity were the variables selected by stepwise discriminant function analysis as the best discriminators between SX and SY. In the second experiment, the sperm nuclear morphometric results from CASA-Morph in nonsexed (mixed SX and SY) and sexed (SX) semen samples from four bulls were compared. FISH allowed a successful classification of spermatozoa according to their sex chromosome content. X-sexed spermatozoa displayed a larger size and fluorescence intensity than nonsexed spermatozoa (P < 0.05). We conclude that the CASA-Morph fluorescence-based method has the potential to find differences between X- and Y-chromosome-bearing spermatozoa in bovine species although more studies are needed to increase the precision of sex determination by this technique.

Tris buffer improves fluorescence yield of ram spermatozoa when evaluating membrane integrity.

This study was designed to evaluate the effect of various buffers on the fluorescence signal intensity of two fluorochromes (IP and CFDA) when used to assess the membrane integrity of ram sperm. Second ejaculates (18) from nine adult males were collected using an artificial vagina and diluted in either MOPS, TRIS, TES, HEPES, citrate, or phosphate-based extenders. Semen samples were stored at 15°C and the membrane integrity was assessed within the first 24 h of storage. Mean fluorescence intensity (FI) of PI- and CDFA-labeled sperm heads and fluorescence background noise (FBN) were determined quantitatively using Image J software. Fluorescence contrast (FC) was expressed as the difference between FI and FBN. Significantly, higher FI and FC were recorded when TRIS diluent was used, rather than the other diluents, both in the propidium- and fluorescein-labeled cells. The citrate and phosphate-based extenders showed intermediate results of FC between those of TRIS and zwitterionic (MOPS, TES and HEPES) groups for the PI-labeled sperm. However, in the CFDA-labeled sperm, the lower values of FC were obtained in the citrate and phosphate groups due to increased levels of FBN. For the membrane-damaged sperm, fluorescent labeling was limited to the sperm heads when TRIS-buffer was used, whereas in the other groups, the sperm tail was also frequently observed. It was concluded that TRIS buffer solution markedly increases the fluorescence yield of IP/CFDA-labeled sperm cells in the ram and that this should be considered when evaluating their membrane integrity.