CASA-Mot in mammals: an update.

Sperm motility is one of the most widely used parameters of sperm quality. Computer-aided sperm motility analysis (CASA-Mot) systems were developed to reduce the subjectivity of sperm motility assessment, and have had broad scientific and practical acceptance. In this review, the sources of variation and current applications of this technology and its relationships with other sperm quality tests are described in detail. Despite remarkable advances in the technique, there is still great need for standardisation in many species, and the numerous factors that affect the results make it difficult to provide universally accepted criteria for classifying semen samples based on sperm motility characteristics. The main fields for CASA-Mot include the study of male fertility and pathologies, evaluation of the effects of physical and chemical agents, improvement of epidemiological survey studies, more precise calculation of seminal doses for farm animals, realisation of basic studies about sperm function, improvement of sperm technologies such as cryopreservation and quality control analysis. Numerous relationships have been established between CASA-Mot and other sperm quality tests, although most of these parameters are complementary. Future CASA-Mot systems will probably be able to integrate several sperm quality parameters with motility.

Determining the relationship between bull sperm kinematic subpopulations and fluorescence groups using an integrated sperm quality analysis technique.

The aim of the present study was to determine whether there is an association between the kinematic sperm subpopulations and fluorescent groups in bulls using a new fluorescent staining method that allows classification of spermatozoa into groups depending on their acrosomal and membrane integrity, as well as functional status, without inhibiting sperm motility. Cryopreserved semen samples from 10 Holstein bulls were used in the study. A multiparametric analysis of results obtained by the ISAS 3Fun kit (Proiser) was performed. The different fluorescent groups were detected and their motility characteristics evaluated using ISAS software. Clustering procedures using the kinematic data resulted in the classification of spermatozoa into three kinematic sperm subpopulations. The distribution of kinematic sperm subpopulations was different between the fluorescent sperm groups (P<0.001), although the correlation between them was low (r=0.113; P<0.01).

Climate factors affecting fertility after cervical insemination during the first months of the breeding season in Rasa Aragonesa ewes.

This study was carried out to examine the impact of several climate variables on the pregnancy rate after cervical artificial insemination (AI) of Rasa Aragonesa ewes. Data were derived from 8,977 inseminations in 76 well-managed flocks performed during the first month of the breeding season (July to October). The following data were recorded for each animal: farm, year, month of AI, parity, lambing-treatment interval, inseminating ram, AI technician, and climatic variables such as mean, maximum and minimum temperature, mean and maximum relative humidity, rainfall, and mean and maximum temperature-humidity index (THI) for each day from day 12 before AI to day 14 post-AI. Means were furthermore calculated for the following periods around AI (day 0): -12 to 0, -2 to 0, AI day, 0 to 2, and 0 to 14. Logistic regression analysis indicated that the likelihood of pregnancy decreased when maximum temperature in the 2 days prior to AI was higher than 30 °C (by a factor of 0.81). Fertility was also lower for primiparous ewes and in multiparous ewes with more than five previous parturitions. Other factors with significant impact on fertility were flock, technician, inseminating ram, and a lambing-AI interval longer than 240 days. It was concluded that the 2 days prior to AI seems to be the period when heat stress had the greatest impact on pregnancy rate in Rasa Aragonesa ewes.

Use of Relief Contrast(®) objective to improve sperm morphometric analysis by Isas(®) casa system in the ram.

The aim of this study was to develop a new method for morphometric assessment of the sperm head and acrosome in the ram. Ejaculates from 10 adult males were collected using an artificial vagina. For each ejaculate, 10 semen smears were prepared, air-dried and divided (in pairs) into the following five treatment groups: (i) washed in distilled water and allowed to dry without further processing (DRY); (ii) fixed in 50% methanol (MET); (iii) fixed in 2% glutaraldehyde (GLUT); (iv) fixed and stained with Hemacolor(®) (HEM) and (v) fixed and stained with SpermBlue(®) (SB). The prepared slides were examined with a 40 × Relief Contrast(®) objective (RC) and processed with ISAS(®) commercial software. The use of RC optics increased the contrast between acrosome and sperm head, allowing capture and morphometric analysis by ISAS of sperm heads and the acrosome, even in non-stained samples. MET and GLUT groups resulted in a lower number of acceptable, that is, correctly delineated, sperm heads than those in the SB, and SB and HEM groups, respectively (p < 0.05). The higher proportion of sperm discarded in MET and GLUT groups may be explained by a higher presence of artefacts. For the majority of the primary morphometric parameters of the sperm head and the acrosomal area, the relationship between treatments was the following: GLUT> HEM≥ MET≥ SB> DRY. When studying the proportion of the sperm head covered by the acrosome, the relation between treatments was: MET> DRY = GLUT = SB> HEM. It was concluded that the new method for sperm morphometric assessment allows the simultaneous assessment of sperm head and acrosome in the ram by the first time, even in unprocessed semen smears.

Comparison of membrane-permeant fluorescent probes for sperm viability assessment in the ram.

This study was designed to compare the effectiveness and usability of four permeant fluorochromes (CFDA; SYBR-14; Hoechst-33342; and acridine orange), combined with propidium iodide to assess sperm membrane integrity. Three different experiments were conducted. The first trial was designed to study the optimal dye concentration and minimum incubation time required to achieve optimum fluorescence intensities and contrast for each fluorochrome combination using ram fresh semen samples. Both SYBR-14 and acridine orange allowed a direct assessment of sperm membrane integrity, without the need of incubating samples, whereas a minimum of 4 and 6 min of incubation at 37°C was necessary to achieve optimum fluorescence intensities in the CFDA and Hoechst groups, respectively. In the second trial, fresh semen samples were mixed with different volumes of membrane-affected sperm (semen treated with three cycles of freezing to -20°C and thawing at room temperature) to produce semen samples with known proportions of damaged spermatozoa. The results were compared with the theoretical values predicted on the basis of the estimations made on fresh and frozen samples. The proportions of damaged sperm in each sample determined using the four fluorochrome combinations agreed with the predicted theoretical values, with the acridine orange/propidium iodide providing the best adjustment. The third experiment was performed to compare the results of sperm membrane integrity using the four fluorochrome combinations. The proportions of plasmalemma-intact sperm determined by acridine orange and SYBR-14 were greater (p < 0.0001) than the proportions of intact sperm determined by CFDA and Hoechst stains. It was concluded that the most efficient combinations to be used in ram sperm were AO/PI and SYBR/PI because it allowed a direct assessment of sperm viability without the need to incubate samples and obtaining reliable results.

Therapeutic approaches to pregnancy loss of non-infectious cause during the late embryonic/early foetal period in dairy cattle. A review.

The problem of pregnancy loss during the late embryonic/early foetal period due to non-infectious causes in dairy cattle is on the rise, especially in high milk producing cows. This review discusses recent therapeutic approaches designed to reduce the incidence of early foetal loss. These strategies are based on the fact that low plasma concentrations of progesterone are commonly related to foetal loss. Progesterone supplementation during the late embryonic/early foetal period has proven useful in curtailing losses, mainly in single pregnancies, whereas a more positive effect of treatment with GnRH than progesterone has been found in twin pregnancies. Therapeutics is not necessary in cows with an additional corpus luteum. In practical terms, suggestions include recording the number of embryos and corpora lutea on first pregnancy diagnosis and checking for normal pregnancy progression on day 60 post-insemination.

Effects of a progesterone-based oestrous synchronization protocol in 51- to 57-day postpartum high-producing dairy cows.

The aim of this study was to investigate the effect of applying a progesterone-based oestrous synchronization protocol at 51-57 days postpartum in high-producing dairy cows. The data analysed were derived from 1345 lactating cows. Cows between 51 and 57 days postpartum were assigned to the groups: control, PRID (receiving a progesterone-releasing intravaginal device for 9 days, and prostaglandin F(2α) 24 h before PRID removal) or GnRH-PRID (the same as the PRID group plus GnRH at PRID insertion). Oestrus was detected by using pedometers and confirmed by examination of the genital tract at AI. Oestrous and conception rates before days 71-77 postpartum, pregnancy loss in early pregnant cows or the cumulative conception rate registered on day 120 postpartum were considered as the dependent variables in four consecutive logistic regression analyses. Based on the odds ratios, the oestrous rate increased by a factor of 1.73 in cows showing oestrus before treatment for each unit increase in the number of previous oestruses; decreased by a factor of 0.44 in the control group with respect to the treatment groups; and by a factor of 0.61 in cows without luteal structures at treatment with respect to cows with corpora lutea. The conception rates of cows inseminated before days 71-77 postpartum remained similar across the groups, whereas the likelihood of pregnancy loss for cows becoming pregnant during this period was 0.11 times lower in the PRID group than in the control. Based on the odds ratio, the likelihood of a higher cumulative conception rate on day 120 postpartum: increased in cows showing oestrus before treatment by a factor of 1.41 for each unit increase in the number of previous oestruses, was reduced 0.56-fold in control cows compared with treated cows, and was also reduced by a factor of 0.98 for each kilogram of milk production increase recorded at treatment. In conclusion, although oestrous synchronization programmes performed in this study did not improve fertility, cows treated with progesterone could be inseminated earlier than untreated cows, such that the treatments increased the cumulative pregnancy rates determined on day 120 postpartum. In addition, fewer pregnancy losses were observed in early pregnant cows in the PRID group than the GnRH-PRID group.

Some factors affecting the abortion rate in dairy herds with high incidence of Neospora-associated abortions are different in cows and heifers.

The aim of this study was to determine if the factors affecting the abortion rate in dairy herds with high incidence of Neospora-associated abortions are different in pregnancies of cows and heifers chronically infected with Neospora caninum. In heifers (n = 229), an increase in the cumulative number of days with a mean relative humidity (RH) lower than 60% during the second trimester of gestation increases the risk of abortion. Yet, the likelihood of abortion was 7.6 times lower for pregnant heifers inseminated with Limousin bull semen, compared with those inseminated with Holstein-Friesian bull semen. In pregnancies of parous cows (n = 521), an increase in rainfall and in the cumulative number of days with a mean RH lower than 60% during the second trimester of gestation increased the abortion rate. However, in contrast, an increase in the lactation number produced a decrease in the abortion rate, with a likelihood of abortion 4.8 times lower for pregnant cows inseminated with Limousin bull semen, and three times lower for those inseminated with Belgian Blue bull semen, compared with dairy cows inseminated with Holstein-Friesian bull semen. Finally, the likelihood of abortion was 3.2 times lower for pregnancies of parous cows with low antibody titres against N. caninum (6-30 units) as compared to those with high antibody titres (>/=30 units), whereas in heifers this variable had no effect. The practical recommendations of the present study include the control of the cow environment during the second trimester of gestation, the priority of culling for parous cows with higher antibody titres against N. caninum and the insemination of Neospora-seropositive cows with semen from the Limousin breed.

Pregnancy patterns during the early fetal period in high producing dairy cows treated with GnRH or progesterone.

In order to explore pregnancy patterns in high producing dairy cows treated with GnRH or progesterone at pregnancy diagnosis (Days 28-34), two consecutive experiments were designed. In Experiment 1, cows bearing a single embryo were randomly assigned to a PRID (n=40; cows fitted with a progesterone releasing intra-vaginal device for 28 days), GnRH (n=40; cows receiving GnRH) or Control (n=26; untreated cows) group. PRID treatment led to a rise in plasma progesterone concentrations in the 7 days following the onset of treatment compared to the other two groups. In Experiment 2, in which we also examined twin pregnancies, animals were randomly assigned to PRID (n=312) or GnRH (n=294) treatment groups. Treatments were the same as described for Experiment 1. Logistic regression procedures revealed that in cows with a single corpus luteum, the probability of pregnancy loss between the first (Days 28-34) and second (Days 65-62) pregnancy diagnosis decreased by a factor of 0.51 in the PRID group compared to the GnRH group. However, in cows with two or more corpora lutea, PRID treatment increased the likelihood of pregnancy loss by a factor of three, compared to GnRH treatment. In cows carrying twins, the conceptus reduction rate was higher (P=0.02) for the GnRH (36%) than for the PRID (16.4%) group. Formation of a new corpus luteum was recorded in 17.7% of cows in the GnRH group. Our results indicate that compared to GnRH treatment, progesterone treatment given at pregnancy diagnosis in high producing dairy cows, reduced by a factor of 0.51 and increased by a factor of 3 the probability of pregnancy loss in cows with a single or with two or more corpora lutea, respectively, and reduced the conceptus reduction rate in cows carrying twins. The practical implications of our findings are that in herds with a high incidence of early fetal loss of a non-infectious nature, treatment at the time of pregnancy diagnosis with PRID in cows with one corpus luteum and with GnRH in cows with two or more corpora lutea should offer considerable benefits.

Factors affecting plasma pregnancy-associated glycoprotein 1 concentrations throughout gestation in high-producing dairy cows.

This study was designed to establish the factors, if any, which could affect plasma pregnancy-associated glycoprotein-1 (PAG-1) expression in a study population of 87 pregnant, high-producing dairy cows. The factors examined were: semen providing breed (Holstein-Friesian vs Limousin), outcome of gestation (male vs female newborn, and singleton vs twin pregnancies), lactation number, milk production at pregnancy diagnosis, plasma progesterone concentration, season of gestation (warm period, March-November vs cool period, December-February), and day of gestation (40, 90, 120, 150, 180 and 210). Pregnancy was diagnosed by transrectal ultrasound on day 40 post-insemination and by palpation per rectum on days 90, 120, 150, 180 and 210. Blood samples were collected from each animal immediately before each pregnancy diagnosis. The relative contributions of the different factors on PAG-1 concentrations were evaluated by GLM repeated measures analysis of variance. No significant effects of the herd, foetal sex, milk production, lactation number and plasma progesterone concentrations were observed. In contrast, twin pregnancy, the use of Limousin semen and conception during the cool period were correlated with significantly increased plasma PAG-1 concentrations throughout gestation. Our data indicate that both cow well-being during early placental development, determined in our conditions by reduced heat stress when conception occurred in the cool season, and crossbreed pregnancies lead to improved PAG-1 production throughout the gestation period.

Anomalous pregnancies during late embryonic/early foetal period in high producing dairy cows.

This study analyses anomalous cases of gestation ending in pregnancy loss during the early foetal period and their effect on progesterone and plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations. Data derived from a large-scale ultrasound pregnancy diagnosis programme in high producing dairy cows. Over a 3-year period (2004-2007), a very low incidence (0.5%: 15 of 3094) of anomalous pregnancies was recorded. The results revealed that the following anomalies were detected on days 35-41 of gestation in cows carrying singletons with one single corpus luteum: embryo death in eight cows (0.3%); and embryo in the uterine horn contralateral to the corpus luteum in seven cows (0.2%). All these animals suffered pregnancy loss during the early foetal period. In cows carrying dead embryos, no signs of conceptus degeneration were observed on pregnancy diagnosis. Amnion size (approximately 25 mm diameter) and uterine horn fluid contents were estimated to be similar to those of the normal pregnant cows in this period. In the contralateral gestations, live embryos were observed in all ultrasound checks before pregnancy loss. Uterine fluid contents increased in the two cows in which gestation continued for more than a week. In the cases of embryo death but not in those of contralateral gestation, a drop in PAG-1 levels was noted prior to pregnancy loss. Two cows carrying dead embryos increased with time allantoic fluid contents. The PAG-1 values increased with time in one cow bearing a dead embryo (from 2.31 to 6.79 ng/ml) and in two of the contralateral gestations (from 1.66 to 2.33 ng/ml and from 0.39 to 6.79 ng/ml, respectively). Results of this study indicate that the foetal membranes continue to undergo some activity following embryo death, and that contralateral pregnancy may determine failure of the gestation process.

Use of image analysis to assess the plasma membrane integrity of ram spermatozoa in different diluents.

Sperm membrane integrity can be assessed by examining a large number of fluorochrome-stained sperm cells over a relative short period of time by flow cytometry or fluorimetry. However, many small laboratories lack a flow-cytometer or fluorimeter for sperm analysis. This study was designed to develop a new image analysis method to evaluate the membrane integrity of ram spermatozoa with the aid of open software, and was divided into three experiments. In the first experiment, the new computer-assisted method was validated by mixing fresh semen samples with different volumes of killed semen in order to know the proportions of damaged spermatozoa in the samples. In the second trial, the new method was compared with the traditional manual counting, and the effect of three extender media on the suitability of the new developed method was evaluated. In the third experiment, the method proposed was tested by comparing the use of milk-, citrate- or TRIS-based diluents for ram semen preservation at 15 degrees C. In all experiments, semen was assessed for plasma membrane integrity and for percentage of motile and progressive sperm by CASA. In the new computer-assisted method, two images of the sperm cells in a given microscopy field are captured and the number of total- and membrane-damaged cells counted. In the first trial, proportions of damaged sperm cells in each sample determined by the automated procedure agreed closely (r2=0.98, P<0.001) with the predicted theoretical values. In experiment 2, the results of membrane integrity obtained using the new method were highly correlated with those provided by the conventional manual counting after PI-CFDA double staining (r=0.99, P<0.001), and also correlated with sperm motility and progressive motility percentages. Viability was significantly higher after dilution with citrate-, than with Tris-based medium, but similar to PBS (70.32+/-3.93, 55.48+/-5.76 and 65.38+/-3.15, respectively), After 0, 24 and 48h of storage, significantly higher percentages of motile, progressive, and membrane-intact spermatozoa were recorded for the milk than for the Tris extenders. Our results validate the new computer-assisted method for assessing sperm membrane integrity in the sheep, and indicate that the milk extender is less damaging to the sperm of this species than citrate or Tris extenders.

Factors affecting the response to the specific treatment of several forms of clinical anestrus in high producing dairy cows.

This study was designed to examine estrous response rates to the therapeutic treatment of clinical anestrus in high producing dairy cows and to identify the factors that could affect these rates. Cows with silent ovulation (Subestrus group), cystic ovarian disease (Cyst group) or ovarian hypofunction (OH group) were given specific treatment for their disorder. Data were derived from 1764 treatments in cows producing a mean of 45.4 kg of milk upon treatment including: 889 subestrous cows, 367 cystic cows and 508 cows with ovarian hypofunction. Cows showing estrus following treatment exhibited a similar pregnancy rate to cows attaining natural estrus used as reference: 33% (337/1006) and 35% (626/1796), respectively. No significant differences in pregnancy rates were observed among the Subestrus, Cyst and OH groups (34% (196/571), 34% (44/130), 32% (97/305), respectively. Based on the odds ratio, an estrous response for all groups was less likely to occur in cows that had suffered previous anestrus, compared to cows that were anestrous for the first time, whereas the likelihood of an estrous response increased in cows treated after 90 days in milk. Our results indicate that previous anestrus and a late stage of lactation can have a negative and positive effect, respectively, on the estrous response to the specific treatment of clinical anestrus shown by high producing dairy cows. Treatment targeted at each type of clinical anestrus can render similar pregnancy rates to those shown by cows in natural estrus.

Factors affecting plasma progesterone in the early fetal period in high producing dairy cows.

The aim of the present study was to determine whether certain animal- or management related factors could affect plasma progesterone concentrations on Day 42 of gestation in high producing dairy cows. Factors affecting early fetal loss were also analyzed. The study population was comprised of 199 pregnant cows classified as having high (> or =9 ng/ml) or low (<9 ng/ml) plasma progesterone concentrations. Through logistic regression procedures it was determined that, based on the odds ratio, cows with two or more corpora lutea were three times more likely to have high progesterone concentrations than cows with a single corpus luteum. Low producing cows during the warm season were 2.86 times more likely to have high progesterone concentrations than the remaining cows. Primiparous cows with high concentrations (>4 ng/ml) of pregnancy associated glycoprotein-1 (PAG-1) were 2.73 times more likely to have high progesterone concentrations than the remaining cows. Of the 199 pregnancies, 25 (12.6%) suffered early fetal loss: 22/136 (16.2%) during the warm season and 3/63 (4.8%) during the cool season, all in cows without additional corpora lutea. Based upon the odds ratio, cows without an additional corpus luteum were 3.67 times more likely to suffer fetal loss during the warm season than during the cool season. Our results indicate that milk production, the presence of two or more corpora lutea and plasma PAG-1 concentrations can affect plasma progesterone concentrations at the onset of the fetal period. The presence of an additional corpus luteum strongly diminished the risk of early fetal loss during the warm period.

Relationships between milk production, ovarian function and fertility in high-producing dairy herds in north-eastern Spain.

In the dairy industry worldwide, reproductive disorders are a major cause of economic losses and a challenge to scientists and technicians. In recent decades, declining fertility and increasing milk production have been widely reported in dairy cattle. In this article, the relationships between milk production, ovarian disorders and fertility in high-producing dairy herds are briefly described. We carried out a retrospective study of 23 204 lactations included in a reproductive control programme in north-eastern Spain, a geographical area experiencing both warm and cool conditions. The data were collected between 1991 and 2007 and refer to cows first inseminated or examined 45-80 days postpartum in five well-managed, commercial, Holstein-Friesian high-producing dairy herds. Ovarian disorders were classified as ovarian inactivity or hypofunction, cystic ovarian disease, sub-oestrus or silent ovulation and sub-luteal function. Ovarian hypofunction and milk production increased throughout the study period and there was a decrease in the pregnancy rate to first artificial insemination (AI). Cows suffering ovarian hypofunction were efficiently treated using combined progestagen-prostaglandin treatments. The incidence of ovarian cysts showed little variation with time. Treatment of this syndrome may include different GnRH-based treatments or manual rupture. During the last 5 years, sub-oestrus was the predominant dysfunction (42.1%) compared with the cystic (6.3%) and ovarian hypofunction (12%) forms. Response of sub-oestrous cows to treatment with luteolitic agents was usually higher than 60%. Ovarian function and fertility were dramatically impaired during the warm period. However, during the later years of the study, the inclusion of fans and water sprinklers for the warm season appeared to overcome the seasonal effect on fertility.

Dynamics of anti-Neospora caninum antibodies during gestation in chronically infected dairy cows.

The dynamics of antibody production against Neospora caninum during the gestation period was examined in chronically infected dairy cows. Data were obtained from 86 pregnant parous dairy cows, 21 of which had suffered abortion. The cows belonged to two herds in which a diagnosis of N. caninum infection had been previously confirmed in aborted foetuses. Pregnancy diagnosis and blood collection were performed on post-insemination Days 40, 90, 120, 150, 180, 210, and at parturition or until the time of abortion detection. Blood plasma was tested for antibodies against N. caninum using ELISA. The non-aborting cows were divided into two groups according to whether their antibody values in the second half of gestation had increased or not, while aborting cows were classified as those showing an antibody peak before abortion or those not showing a pre-abortion peak. Differences in antibody values throughout pregnancy in each group of non-aborting and aborting cows were analysed by GLM repeated measures of analysis of variance. While 32 non-aborting cows (49%) showed a significant and consistent increase in anti-Neospora antibody values during the second half of gestation, antibody values in the remaining 33 non-aborting cows were practically constant throughout gestation. An antibody peak around abortion was observed in 11 aborting cows (52%), while antibody values in the remaining 10 aborting cows were similar before and at abortion. Seroprevalence fluctuations, defined as seronegative blood samples at some point during the gestation period, were, furthermore, observed in 2 aborting and 11 non-aborting cows. Our results indicate two clearly distinguishable types of humoral immune dynamics throughout gestation: an increased or flat production of antibodies during the second half of gestation in non-aborting animals and before abortion in aborting cows. The observation that some Neospora-infected dams can exhibit negative antibody values at any time during gestation, particularly at parturition or abortion, prompts future studies designed to explore the use of new ELISA strategies at the farm level.

Progesterone supplementation during mid-gestation increases the risk of abortion in Neospora-infected dairy cows with high antibody titres.

Progesterone has been reported to reduce Th1 responses during pregnancy. Thus, given that an excessive Th1 response could lead to abortion in Neospora-infected dairy cows, we hypothesized a reduction in abortion risk following progesterone supplementation during the mid-gestation period in these animals. Seropositive animals were assigned on day 120 of gestation to a Control (n=33) or Treatment (n=34) group. Treatment consisted of progesterone supplementation by fitting a progesterone releasing intravaginal device containing 1.55g of progesterone for 28 days. Mean Neospora caninum antibody titres tested by ELISA on days 40, 90 and 120 of gestation were used to classify the cows as showing high (> or =30 units) or low (<30 units) titration. Abortion rate showed a significant increase (P<0.01) in the Treatment group (35%) compared to the Control (9%). Using logistic regression procedures, there was a significant (P<0.0001) interaction between progesterone treatment and antibody titration. Progesterone-treated cows with a high antibody titre showed a likelihood of abortion 14.3 times higher than those not treated, while in cows with low antibody titres, exogenous progesterone had not effect. Our results do not support our starting hypothesis and suggest that progesterone supplementation in cows with high N. caninum antibody titres increases the risk of abortion possibly by affecting cell-mediated immune responses.

Milk production correlates negatively with plasma levels of pregnancy-associated glycoprotein (PAG) during the early fetal period in high producing dairy cows with live fetuses.

This study was designed to establish possible factors affecting plasma pregnancy-associated glycoprotein (PAG) concentrations during early pregnancy in high producing dairy cows with live fetuses. Blood samples were obtained on days 35, 42, 49, 56 and 63 of gestation from 80 lactating cows in two herds carrying live fetuses. Radioimmunoassay systems were used to determine PAG (RIA-497 and RIA-706) and progesterone concentrations. We evaluated the effects on PAG concentrations of herd, lactation number, sire of fetus, day of gestation, fetus number, plasma progesterone and milk production at each time point established, along with possible paired interactions. Mean milk production per cow approached 41kg during the study period. PAG concentrations were not affected by herd, lactation number or plasma progesterone concentration. Significant positive effects on PAG concentrations were shown by the gestation day, and the interaction between day of gestation and twin pregnancy. Significant differences between bulls and a significant negative correlation between milk production and PAG values on day 63 of pregnancy were also detected. Proportions of blood samples showing undetectable PAG levels and false negative diagnoses throughout the study period were significantly higher (P<0.001) using the RIA-497 system (2.5% and 5.3%, respectively) compared to RIA-706 (0% and 0.8%, respectively). Our findings suggest that PAG concentrations during the early fetal period are related to the day of gestation, milk production, number of fetuses and sire of fetus in high producing dairy cows. Under our working conditions, the RIA-706 method was better at detecting plasma PAG molecules than the RIA-497 system.

Factors affecting the fertility of high producing dairy herds in northeastern Spain.

Infertility has been often correlated to a rising milk yield in high producing dairy cattle. The aim of the present study was to evaluate, using logistic regression procedures, the effects of several management indicators on the fertility of four dairy herds in northeastern Spain. Data derived from 10,965 artificial insemination (AI). The factors examined were: herd, milking frequency (three versus two milkings per day), lactation number, previous twinning and disorders such as placenta retention and pyometra, milk production at AI, the inseminating bull, season (warm versus cool period) and year effects, AI technician and repeat breeding syndrome (cows undergoing four or more AI). Our findings indicated no effects on fertility of the herd, year of AI, previous twining, placenta retention and pyometra and milk production at AI. Based on the odds ratios, the likelihood of pregnancy decreased: in cows milked three times per day (by a factor of 0.62); for each one unit increase in lactation number (by a factor of 0.92); for inseminations performed during the warm period (by a factor of 0.67); in repeat breeder cows (by a factor of 0.73); and when 3 of the 45 inseminating bulls included in the study were used (by factors of 0.35, 0.43 and 0.44, respectively). Of the 13 AI technicians participating in the study, 3 were related to a fertility rate improved by odds ratios of 1.86, 1.84 and 1.30, respectively, whereas 2 technicians gave rise to fertility rates reduced by odds ratios of 0.64 and 0.49, respectively. Under our study conditions, management practices were able to compensate for the effects of previous twining and reproductive disorders such as placenta retention and pyometra. However, fertility was significantly affected by the factors milking frequency, AI technician, inseminating bull, repeat breeding syndrome, lactation number and AI season.

Protection against abortion linked to gamma interferon production in pregnant dairy cows naturally infected with Neospora caninum.

Many immunological aspects of pregnancy, such as the role played by gamma interferon (IFN-gamma) in abortion, are not well understood. Neospora caninum is an intracellular protozoan considered to be among the main causes of abortion in cattle worldwide. The present study analyzes the interaction between IFN-gamma production and N. caninum infection in naturally infected pregnant cows. Data were obtained from 126 pregnant cows: 86 seropositive and 40 seronegative for the parasite. Pregnancy diagnosis and blood sample collection were performed on days 40, 90, 120, 150, 180 and 210 post-insemination or until the time of abortion detection. Plasma was tested for antibodies against N. caninum and IFN-gamma. Interferon-gamma was detected at some point along the pregnancy in 16 (19%) of the 86 Neospora-seropositive cows yet was undetectable in the 40 seronegative animals. Of the 126 pregnancies examined, 22 (17.5%) ended in abortion. Abortion occurred in 24.4% of seropositive cows (21/86) and in 2.5% of seronegative animals (1/40). Significant (P<0.0001) interaction was observed between Neospora-seropositivity and IFN-gamma production. Based on the odds ratio, the risk of abortion was 15.6 times higher in seropositive cows not producing IFN-gamma than in seronegative animals, whereas neosporosis had no effect in seropositive cows with IFN-gamma production. A significant (P=0.001) negative effect of IFN-gamma production on the Neospora titer was furthermore observed in the 65 non-aborting seropositive animals. These results indicate that IFN-gamma production affords protection against abortion in Neospora-infected cows and also point to a reduced humoral immune response to N. caninum during gestation in cows producing IFN-gamma.