Early prediction of oestrus for herd fertility management in cattle and buffaloes - a review.

Oestrus is defined as a period when a female animal exhibits characteristic sexual behaviour in the presence of a mature male. Oestrous manifestation in dairy animals is due to the oestrogen (E2) effect on the central nervous system (CNS). It is a critical issue to be considered on a priority basis. Inefficient oestrous detection reduces the fertility status of the herd. The primary and most reliable indicator of oestrus is standing to be mounted by a bull or another female herd mate, signalling receptivity and the pre-ovulatory state in dairy cattle. Oestrous detection is primarily a management challenge requiring skill and vigilance. To improve the efficiency of oestrous detection in dairy cattle, visual observation is one of the best methods if done three times a day; however, heat detection aids, if combined, give better results. However, techniques like using teaser bulls, tail painting, chin ball markers, ultrasound (USG) examination, hormonal analysis and examination of cervicovaginal mucus (CVM) improve oestrous detection efficiency. Moreover, the changes in production systems have reduced the expression of oestrous behaviour among cows, due to higher oestrogen (E2) metabolism. Therefore, automated systems, such as pedometers, accelerometers and acoustic sensors like infrared thermography (IRT) and image processing, have significantly enhanced reproductive performance by facilitating oestrous detection and optimizing insemination schedules. From this review, we would conclude that oestrous detection alone contributes considerably to the reproductive status of the herd; therefore, applying different methods of oestrous detection reduces the incidence of missed oestrus and improves the fertility status of the herd.

Evaluation of antibacterial oxygen/ozone mixture in vitro activity on bacteria isolated from cervico-vaginal mucus of cows with acute metritis.

Ozone is an oxidating gas showing a strong microbicidal activity on bacteria, fungi, viruses and protozoa. The aim of this study was to test the in vitro bacteriocidal action of an Ozone/Oxygen gas mixture on bacteria isolated from the cervico-vaginal mucus of cows affected by acute metritis. A pilot study was initially carried out on reference strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Mycoplasma bovigenitalium ATCC 19852) that were tested with three different treatments: a control positive baseline group (B-group) was incubated without gas treatment, a control positive oxygen group (O2-group) was treated with pure oxygen 100%, and the treated group (T-group) was exposed to a gaseous constant flow of an Ozone/Oxygen mixture, at 50, 35, 20 µg Ozone/ml and for 5, 3 and 1 min for every different Ozone concentration. In both positive control groups, the number of colony forming units (CFU) per ml was higher than 300 CFU/ml (E. coli and S. aureus) and higher than 30 CFU/ml for M. bovigenitalium, after incubation. The T-groups showed a minimal bacterial growth equal to or lower than 1 CFU/ml per plate. Based on the results of the pilot study, a second phase was performed on bacteria isolated from the cervico-vaginal mucus (Klebsiella pneumoniae, Enterobacter agglomerans, E. coli, Proteus mirabilis and M. bovigenitalium) using the lower concentration of 20 µg/ml of Ozone for the minimum exposure time of 1 min. The E. coli and S. aureus reference strains and the clinical isolates (K. pneumoniae, E. agglomerans, E. coli, P. mirabilis) were incubated at 37 °C for 48 h and the colonies were manually counted at 24 h and 48 h following inoculation. The cultures of M. bovigenitalium (both ATCC and clinical isolate) were incubated in a jar with modified atmosphere conditions with 5% CO2 at 37 °C for 4-7 days and colony counting was performed. The second phase showed a low number of CFUs (equal to or less than 7 CFU/ml) for the clinical isolates K. pneumoniae, E. agglomerans, E. coli and P. mirabilis, and, of note, for M. bovigenitalium, both ATCC and clinical isolate, the growth was completely inhibited. Ozone was demonstrated to have a bacteriocidal activity. This study encourages further research of in vivo application of low doses of gaseous Ozone for the treatment of metritis in cows by using minimal exposure times.

Staphylococcus pasteuri (BCVME2) Resident in Buffalo Cervical Vaginal Mucus: A Potential Source of Estrus-Specific Sex Pheromone(s).

Mammals have microbes resident in their reproductive tract, some of which can be pathogenic while others may play a role in protecting the tract from infection. Volatile compounds play a role as sex pheromones that attract males for coitus during female estrus or heat. It is likely that these compounds themselves are secondary metabolites of bacterial flora resident in the vagina. In order to substantiate this hypothesis, bacteria were isolated from cervico-vaginal mucus (CVM) of buffalo during various phases of the estrous cycle and identified, using morphological, biochemical and molecular characteristics, as Bacillus during preestrus and diestrus, and as Staphylococcus during all three phases of the estrous cycle. Populations of Staphylococcus differed between different phases of the estrous cycle, the predominant forms being S. warneri (BCVMPE1_1) during preestrus, S. pastueri (BCVME2) during estrus and S. epidermis (BCVMDE3) during diestrus. Mice were used as chemosensors to differentiate the estrus-specific S. pasteuri (BCVME2) from the others. Chemical analysis showed that S. pasteuri (BCVME2) produced acetic, propanoic, isobutyric, butyric, isovaleric and valeric acids. In addition, it was shown that S. pasteuri (BCVME2) volatiles influenced the sexual behaviors, flehmen and mounting, of the bull. Thus, S. pasteuri (BCVME2) is a potential source of vaginal pheromone(s) during estrus in buffalo.

A simple and reliable refractometric method to determine the total solids concentration of the cervico-vaginal bovine mucus samples.

Cervico-vaginal mucus (CVM) is a viscoelastic substance continuously produced by secretory cells of the endocervix and the vagina of cows. Its physicochemical composition varies depending on the hormonal status of the estrous cycle. In veterinary medicine refractometry is a widely diffused technique to determine total solids (TS) content of biological samples, but there are not published data of CVM total solids from refractometric measures. Refractometric TS determination contributes to the qualitative constituents analysis of CVM, additionally it is an easier and more inexpensive technique than gravimetric TS determination. The main goal of the present paper was to validate a refractometric method to estimate TS concentration of the soluble fraction of CVM samples. Samples were collected from seventy-three Holando Argentino cows of Santa Fe province farms in Argentina. Cows were classified in three experimental groups: healthy, subclinical (SE) and clinical endometritis (CE) group. To achieve a solubilisation protocol for CVM samples, four Triton™ X-100 concentrations were tested. Refractive index (RI) and gravimetric total solid (gTS) concentration of solubilised samples were determined for the three experimental groups. A mathematical equation was determined with the experimental data from the healthy group, in order to obtain calculated total solid concentration (cTS) from refractivity (R) values. To validate the RI method for CVM samples, cTS concentrations were compared with gTS concentrations from endometritis group samples. Triton™ X-100 0.01% (V/V) improved CVM samples handling and did not change physicochemical parameters (gTS, Na+ and K+ concentration, and RI values). The linear regression equation obtained was: cTS (g/dL) = (R - 0.67)/16.2, r2 = 0.91. Correlation between gTS and cTS concentration was: r = 0.97 for SE group and r = 0.97 for CE group. The homogenization protocol allowed the measurement of physicochemical parameters without altering their values. A high correlation coefficient between cTS and gTS postulates refractometry as an accurate method to determine TS concentration for solubilised CVM samples.

Significance of cervico-vaginal microbes in bovine reproduction and pheromone production - A hypothetical review.

The vaginal microbiota has been studied in animal reproduction and fertility, in particular little information of vaginal microbes in reference to bovine reproduction and pheromone production is known. The vaginal mucosa in healthy cow is colonized by an equilibrated and dynamic composition of aerobic, facultative anaerobic and obligate anaerobic microbes. Cervico-vaginal mucus (CVM) composition, viscosity and volume vary with the cyclicity and health status of the reproductive tract. In addition, CVM contains pheromones, volatile compounds, and proteins that attract males for coitus. Commensal microbiota plays a key role in protection of the genital tract from pathogenic microbes by competition effect. In the bovine species, the microbial composition, its abundance and diversity in the female gut, vagina, urine, saliva, and feces, and the associated chemical communication remains poorly documented. The impact of microbes in the reproductive tract of cow, buffalo and certain mammals are discussed in this review. Since the microbial population diversity of CVM is modified during estrus phase it presumes that it may have a role for pheromone production in conspecific. Herein, we would like to critically discuss the current state of knowledge on microbially produced signals in animals and the role of genital and CVM microbiota in estrous cycle and pregnancy.

Molecular biology as a diagnostic tool for detection of Leptospira spp. in cows of a border region ­ case report

The reproductive efficiency of livestock is the basis for the success of livestock, dairy or beef, and having high reproductive performance depends on several factors within the production system and the presence of infectious diseases of the reproductive sphere in the herd is one of the factors that can compromise that efficiency. The aim of this study was to use molecular biology as a diagnostic tool for the detection of Leptospira spp. DNA in cows with reproductive disorders on a rural property in the municipality of Boca do Acre, Amazonas, Brazil. Vaginal mucus was collected from nine Nelore breeding cows with a history of abortion and birth of weak calves submitted to DNA extraction and nested-PCR technique for 16S gene amplification at the bacterial genus level. Of the nine samples analyzed, five (55.55%) amplified a product of 331bp. The municipality of Boca do Acre is bordered by Peru and Bolivia, and knowledge of the prevalence of the disease, serovars, and circulating Leptospira species is essential for the adoption of measures related to animal husbandry, as well as health education for ranchers and their workers to avoid a possible occupational infection since this disease is considered an important zoonosis. New molecular studies using primers that allow the identification of the Leptospira species and mainly pathogenic species should be conducted in this region in order to elucidate the possible species of this etiological agent and the possible reservoirs of the disease to begin the understanding of the epidemiology of this disease in cattle in this region of border.

Investigation of vaginal mucus parameters: Development of models for staging the oestrous cycle of the Bunaji cow.

Changes in some vaginal mucus parameters were studied in order to generate predictive models capable of enhancing oestrous cycle staging, using equal groups (unsynchronized-USC [no treatment] and synchronized-SC [Synchromate® i/m on d0, d11]) of Bunaji cows (n = 48) aged 3-4 years. Vaginal mucus was collected (starting d11 in SC) daily over 26 days using standard procedures. Physical (viscosity, elasticity, density, resistivity) and biochemical (pH, glucose, cholesterol, total protein, calcium, magnesium, sodium, potassium) parameters were evaluated using standard procedures. Data were analysed using chi-square and multinomial logit regression modelling. Models generated using oestrus as reference categories were ascertained for accuracies. Chi-square values for viscosity, elasticity and density were significant (p < .01) in USC and SC across stages of the cycle. Results for USC showed that pH and cholesterol were predictive (p < .01) for pro-oestrus, metoestrus and dioestrus, while total protein was predictive (p < .01) for dioestrus only. Similarly, magnesium was predictive (p < .05) for pro-oestrus. For SC, pH, magnesium and cholesterol were predictive (p < .01) for pro-oestrus, metoestrus and dioestrus, while total protein was predictive (p < .01) for pro-oestrus and dioestrus. Potassium and total protein were also predictive for metoestrus at 10% and 5% significance levels, respectively. Though findings suggest the usefulness of magnesium in staging the oestrous cycle only in synchronized cows, pH, total protein and cholesterol appeared to be the more important vaginal mucus parameters in Bunaji cows, regardless synchronization. Furthermore, the models developed showed high accuracy levels for staging the oestrous cycle in USC (100%) and SC (89%).

Optimal timing of artificial insemination and changes in vaginal mucous characteristics relative to the onset of standing estrus in Beetal goats.

The current study was conducted to determine the optimal timing of artificial insemination and vaginal mucous characteristics relative to the onset of estrus in goats. Does (n = 257) were estrous synchronized using hormonal treatments. Intracervical inseminations with fresh semen were performed at 0, 12, 24 and 36 h after the onset of estrus. Characteristics of vaginal mucus (i.e., color, consistency, and volume) were observed and graded at the time of AI to calculate cumulative mucous score. The vaginal electrical resistance (VER) was recorded (Draminski® detector) at the time of AI. Pregnancy rate was less (P < 0.05) when inseminations occurred at 0 h (28.6%) compared with 12 (58.3%) and 24 h (56.4%) after the onset of estrus; however, pregnancy rate at 36 h (54.5%) did not differ (P> 0.05) compared with inseminations at 0, 12 or 24 h after estrous onset. Relative odds for pregnancy rate at 12, 24 and 36 h were 5.24, 5.20 and 3.29 times greater compared with 0 h. Cumulative mucous score varied (P < 0.05) relative to the onset of estrus and correlated well (P < 0.05) with the color and consistency than volume of the mucus. The VER was less (P < 0.05) at 12 than 0 and 36 h after estrous onset. In conclusion, goats can be inseminated between 12-36 h after the onset of estrus; however, the chances of pregnancy are greater with inseminations at the 12 or 24 h time periods.

Purulent vaginal discharge diagnosed in pasture-based Holstein-Friesian cows at 21 days postpartum is influenced by previous lactation milk yield and results in diminished fertility.

In a subset of dairy cows, prolonged pathological uterine inflammation results in purulent vaginal discharge (PVD), which can have negative consequences for both fertility and milk production. However, unlike for intensive systems, analysis of the effects of PVD in predominantly pasture-based herds is limited. The objective of this study was to assess the effect of PVD in spring-calving, pasture-based dairy cows on production and reproduction indices, stratified according to previous full-lactation milk yield. We assessed clinical disease as defined by vaginal mucus score (VMS) in 440 Holstein-Friesian cows from 5 farms. Cows were categorized as healthy (VMS 0) or having PVD (VMS 1-3) at 21 d postpartum. We recorded 305-d milk, milk protein, and milk fat yields (kg) before and after disease diagnosis, as well as fertility data, such as services per conception and the calving-conception period (CCP). Using SAS 9.4 (SAS Institute Inc., Cary, NC), we analyzed data using PROC MIXED, PROC PHREG, and PROC LOGISTIC to determine the least squares means differences and hazard and odds ratios between the groups, respectively. Overall, a 60% prevalence of PVD was recorded at 21 d postpartum. Milk yield and milk constituents were similar between all VMS categories and between healthy cows and cows with PVD. Although cows in the 4 VMS categories had statistically similar CCP, cows with PVD had a significantly longer CCP than healthy cows on average (9 d). The hazard ratio for cows with PVD was 0.66, indicating a 34% higher risk of a prolonged CCP than healthy cows. Odds ratio analysis determined that cows with PVD were 3 times more likely not to conceive at all, twice as likely not to conceive at first service, twice as likely not to conceive by 100 d postpartum, and 3 times more likely to fail to conceive before 150 d postpartum compared with healthy cows. Cows were retrospectively categorized as having low or high milk yield, based on whether they were above or below the median 305-d milk yield of the study population (6,571 kg) in the lactation before vaginal mucus scoring. Based on a univariate odds ratio, high-yield cows were 1.6 times more likely to present with PVD in the subsequent lactation. The number of services per conception did not differ between healthy and PVD cows in the low- and high-yield groups. In the high-yield group, cows with PVD were 4.9 times more likely not to conceive, 2.7 times more likely to require multiple services to conceive, 2.1 times more likely to remain not pregnant by 100 d postpartum, and 4.4 times more likely to remain not pregnant by 150 d postpartum. The CCP was also significantly longer in cows with PVD than their healthy counterparts (115.9 ± 4.9 and 104 ± 7.4 d, respectively). In conclusion, PVD significantly increased the CCP in all cows, but to a greater extent in cows with a high milk yield in the lactation before disease diagnosis.

Improved detection of biomarkers in cervico-vaginal mucus (CVM) from postpartum cattle.

BACKGROUND: In the postpartum cow, early diagnosis of uterine disease is currently problematic due to the lack of reliable, non-invasive diagnostic methods. Cervico-vaginal mucus (CVM) is an easy to collect potentially informative source of biomarkers for the diagnosis and prognosis of uterine disease in cows. Here, we report an improved method for processing CVM from postpartum dairy cows for the measurement of immune biomarkers. CVM samples were collected from the vagina using gloved hand during the first two weeks postpartum and processed with buffer alone or buffer containing different concentrations of the reducing agents recommended in standard protocols: Dithiothriotol (DTT) or N-Acetyl-L-Cysteine (NAC). Total protein was measured using the bicinchoninic acid (BCA) assay; interleukin 6 (IL-6), IL-8 and α1-acid glycoprotein (AGP) were measured by ELISA. RESULTS: We found that use of reducing agents to liquefy CVM affects protein yield and the accuracy of biomarker detection. Our improved protocol results in lower protein yields but improved detection of cytokines and chemokines. Using our modified method to measure AGP in CVM we found raised levels of AGP at seven days postpartum in CVM from cows that went on to develop endometritis. CONCLUSION: We conclude that processing CVM without reducing agents improves detection of biomarkers that reflect uterine health in cattle. We propose that measurement of AGP in CVM during the first week postpartum may identify cows at risk of developing clinical endometritis.

Cervico-vaginal mucus (CVM) - an accessible source of immunologically informative biomolecules.

Cervico-vaginal mucus (CVM), the product of epithelial cells lining the uterus, cervix and vagina, is secreted to facilitate uterine lubrication and microbial clearance. Predominantly composed of water and mucins, CVM also contains high levels of immuno-active proteins such as immunoglobulin A (IgA), lactoferrin and lysozyme which protect against infection by blocking adhesion and mediating microbial killing. The repertoire of cytokines, chemokines and antimicrobial peptides is predominantly generated by the secretions of endometrial epithelial cells into the uterine lumen and concentrated in the CVM. The quantity and relative proportions of these inflammatory biomarkers are affected by diverse factors including the estrus cycle and health status of the animal and therefore potentially provide important diagnostic and prognostic indicators. We propose that measuring molecular signatures in bovine CVM could be a useful approach to identifying and monitoring genital tract pathologies in beef and dairy cows.

Profiling inflammatory biomarkers in cervico-vaginal mucus (CVM) postpartum: Potential early indicators of bovine clinical endometritis?

Endometritis significantly impacts fertility and milk yield, thus reducing profitability of the dairy production. In cows that develop endometritis, normal postpartum endometrial inflammation is dysregulated. Here, we propose that endometrial inflammation is reflected in cervico-vaginal mucus (CVM) which could therefore be used as a prognostic tool. CVM was collected from 20 dairy cows (10 with clinical endometritis and 10 healthy) 7 and 21 days postpartum (DPP). Polymorphonuclear (PMN), mononuclear leukocyte and epithelial cells were counted, total protein levels were estimated and levels of IL-1ß, IL-6, IL-8, serum amyloid A (SAA), haptoglobin (Hp) and C5b were analyzed by ELISA in CVM. PMN were consistently high in CVM from 7 to 21 DPP, but were higher in CVM from cows with clinical endometritis 21 DPP compared with healthy cows. In contrast, there were more epithelial cells in healthy cows 21 DPP than in clinical endometritis animals. Total protein levels decreased significantly in CVM from healthy cows between days 7 and 21 postpartum. All inflammatory biomarkers except C5b, remained high in cows with clinical endometritis from 7 to 21 DPP, indicating sustained and chronic endometrial inflammation. IL1, IL-6, IL-8 and Hp levels were higher in CVM from cows with clinical endometritis compared to healthy cows 21 DPP. Interestingly IL-1ß levels were raised in CVM from clinical endometritis but not in healthy cows 7 DPP suggesting that early measurement of IL-1ß levels might provide a useful predictive marker of clinical endometritis. In contrast, SAA and C5b levels were increased in healthy cows 21 DPP, compared to cows with clinical endometritis suggesting that these acute phase proteins might have an anti-inflammatory role. Our results show that CVM is convenient for profiling disease-associated changes in key inflammatory molecules postpartum and reaffirms that sustained inflammation is a key feature of clinical endometritis in the dairy cow.

Endometritis Increases Pro-inflammatory Cytokines in Follicular Fluid and Cervico-vaginal Mucus in the Buffalo Cow.

Emerging evidence shows that some of the pro-inflammatory cytokines are elevated not only in the endometrium but also in the follicular fluid of cows with endometritis. Developing a cervico-vaginal mucus (CVM) based test has the potential for becoming a pen-side test because of the ease of sample collection. The present study describes the results of two different experiments. The first experiment was conducted to investigate the influence of endometritis on the proinflammatory cytokines of follicular fluid based on the reproductive tracts of buffalo collected at a slaughter house Buffalo genitalia were categorized into purulent endometritis (PE), cytological endometritis (CE), and non-endometritis (NE) based on the white-side test and endometrial cytology, respectively (n = 14/group). Each group was subdivided into follicular and mid-luteal stage (n = 7/stage) and the follicular fluid was collected from the largest follicle. Second experiment was done to study the difference in the levels of proinflammatory cytokines in the CVM of repeat breeders with subclinical endometritis presented to the clinic. CVM was collected from the repeaters (n = 10) and non-repeaters (n = 10) through aseptic trans-vaginal aspiration. The pro-inflammatory cytokines such as IL-1ß, IL-6, IL-8, and TNFα were quantitated through bovine specific ELISA kits. Significantly higher concentrations of pro-inflammatory cytokines (IL-1ß, IL-8, IL-6, and TNFα) along with low intra-follicular estradiol in buffaloes of PE and CE groups suggest that endometritis impedes the follicular steroidogenesis. Significantly higher concentration of IL-1ß and TNF-α in the CVM of repeaters indicate their potential as a pen-side diagnostic test for CE.

Investigation of the possible role of Chlamydophila abortus in reproductive failures in nrazilian herds of domestic ruminants

Chlamydophila abortus (C. abortus) infection is related to reproductive failure in domestic ruminants. Although it has not been well characterized worldwide, this pathogen has already been identified in some European countries and in the USA. In Brazil, preliminary studies have shown serological evidence of C. abortus infection in herds with low antibody prevalence. Until now, the identification of C. abortus in biological samples from females presenting reproductive failures has not been described in Brazilian herds of domestic ruminants. The aim of this study was to evaluate the presence of the C. abortus in a collection of abortions from cattle (n=85), sheep (n=12), and goats (n=8), in samples of vaginal mucus from cows (n=13), sheep (n=90), and goats (n­=20), and in semen from sheep (n=10) and goats (n=5). The specimens (n=243) were evaluated using a PCR assay developed to amplify the 16S-23S rRNA intergenic space of C. abortus. A PCR assay with an internal control, which amplifies a fragment from the ND5 gene of bovine mitochondrial DNA, was used in order to evaluate the efficiency of the DNA extraction and of the PCR reaction. All biological samples (n=243) included in this study were negative for C. abortus in the PCR assay. The internal control enabled the amplification of a product from the bovine mitochondrial ND5 gene in all cattle abortion samples (n=85). Given the serological evidence indicating the presence of C. abortus infection in Brazilian herds of domestic ruminants, and considering the wide sampling evaluated, the failure to identify C. abortus in this survey suggests that the frequency of clinical signs in infected animals may be low or even absent.

A infecção pela Chlamydophila abortus (C. abortus) em ruminantes domésticos está relacionada com distúrbios reprodutivos. Apesar de ainda pouco estudada em todo o mundo, a infecção já foi identificada em alguns países europeus e também nos EUA. No Brasil, estudos preliminares demonstraram evidências sorológicas da infecção em alguns rebanhos com baixa prevalência de anticorpos. Até o momento ainda não foi possível a identificação de C. abortus a partir de material biológico proveniente de fêmeas com problemas reprodutivos em rebanhos brasileiros. O objetivo deste trabalho foi avaliar a presença da C. abortus em uma coleção de abortos bovinos (n=85), ovinos (n=12) e caprinos (n=8), em amostras de muco vaginal bovino (n=13), ovino (n=90) e caprino (n=20), e em sêmen ovino (n=10) e caprino (n=5). As amostras biológicas (n=243) foram avaliadas por meio de técnica de PCR desenvolvida para a amplificação do espaço intergênico 16S-23S RNAr da C. abortus. Um controle interno da reação, que amplifica um fragmento do gene ND5 do DNA mitocondrial de bovino, foi utilizado para a avaliação da eficiência da extração e da amplificação do DNA nas amostras provenientes de abortamento bovino. Todas as amostras biológicas (n=243) incluídas nesse estudo resultaram negativas para a C. abortus na PCR. O controle interno da reação possibilitou a amplificação de um produto do gene ND5 mitocondrial bovino em todas as amostras de aborto bovino (n=85). Apesar de evidências,,sorológicas indicarem a presença da infecção por C. abortus em rebanhos de ruminantes no Brasil, e considerando o número de amostras biológicas avaliadas, a não identificação de C. abortus nesse estudo sugere que a frequência de sinais clínicos nos animais infectados pode ser baixa ou mesmo ausente.

Efecto del coumestrol sobre la producción espermática y la conducta de exploración olfatoria de perros estimulados con moco vaginal estral / Effect of coumestrol on sperm production and smell exploratory behavior in dogs stimulated with vaginal mucus

Dog overpopulation is a major health problem in developing countries due to the existence of some zoonotic diseases in which dogs act as reservoirs, besides the aggressive events to humans. Distribution, behavior patterns and combined methodologies are needed aspects in the design of successful dog population control programs. Coumestrol is a phytoestrogen which induces alterations in the reproductive male system, when bind to alpha and beta estrogen receptors acting as an agonist or antagonist fashion. Both receptor types also exist in central nerve regions governing sexual behavior of those animals such as the preoptic area, ventro medial nucleous, the amygdala and the olfactory bulb. In this study, 300 μg/kg coumestrol was orally administered to male dogs, once a week for a 4 week period. Dogs were freed for 5 min in a 9 m² area having a recipient containing vaginal discharges from estrous dog females and other similar vessel containing sterile saline solution. Smelling latency time for each recipient, smelling frequency and territory marking in response to stimulus, was recorded. At the end of the test, semen was collected and evaluated. A significative difference (P < 0.005) in smelling latency time and smelling frequency to the vaginal discharge was found; sperm count decreased from 518.4 ± 215.4 × 10(6) to 57.6 ± 50.4 × 10(6) at week 4 and the abnormal sperm morphology increased from 14.7 ± 3.3% at 0 week to 60.0 ± 20%. In conclusion, 300 μg/kg coumestrol given orally to male dogs for 4 weeks induces alterations in the olfactory behavior along with an oligo and teratospermic effect.

La sobrepoblación canina es un problema importante de salud pública debido a la transmisión de enfermedades zoonóticas y las agresiones hacia el humano. En el diseño de programas para controlar la población canina se requiere del conocimiento de su distribución, comportamiento y metodologías combinadas para tener éxito. El coumestrol es un fitoestrógeno que induce alteraciones en el aparato reproductor de los machos al unirse a los receptores estrogénicos alfa y beta, en donde actúa de manera dosis-dependiente como agonista o antagonista. Estos receptores también existen en las estructuras del sistema nervioso que regulan el comportamiento sexual, como la región preóptica, núcleo ventromedial, la amígdala y el bulbo olfatorio. En este estudio se administró coumestrol (300 μg/kg) por vía oral a perros machos, una vez por semana durante cuatro semanas; los perros se colocaron durante cinco minutos en un área aislada de 9 m² en donde se colocó un frasco conteniendo secreciones vaginales obtenidas de perras en estro y otro con solución salina estéril. Se registró el tiempo de latencia de los perros para olfatear cada frasco, su frecuencia de respuesta y la frecuencia con que se presentó conducta de marcaje en respuesta al estímulo. Una vez concluido el experimento, se obtuvo semen y se evaluó. Se encontró diferencia significativa (P < 0.005) en el periodo de latencia y frecuencia con la que el macho se acercó a oler las secreciones vaginales y el conteo espermático disminuyó de 518.4 ± 215.4 × 10(6) al inicio del estudio a 57.6 ± 50.4 × 10(6) en la semana cuatro y el porcentaje de anormalidades espermáticas aumentó de 14.7 ± 3.3 puntos en la semana 0 a 60.0 ± 20.0% en la semana cuatro. Se concluye que el tratamiento de perros con coumestrol durante cuatro semanas ocasiona alteraciones en la conducta de exploración olfatoria y tiene un efecto oligospérmico y teratospérmico.