Review: Feed residues of glyphosate - potential consequences for livestock health and productivity.

Glyphosate is the active ingredient in a wide range of herbicides used for weed control, including weed control in genetically modified, glyphosate-insensitive crops. In addition, glyphosate herbicides are used for pre-harvest desiccation of glyphosate-sensitive crops. Together, the use of glyphosate leads to residues in livestock feed. In addition to its herbicidal property, glyphosate has documented antimicrobial and mineral-chelating properties. The aim of the present paper is to address, based on the published literature and own observations, whether dietary glyphosate residues may affect livestock gut microbiota and/or mineral status potentially with derived unfavourable effects on animal health and productivity. However, and as reported, literature on the potential effects of glyphosate on livestock is very scarce and mainly reporting in vitro studies; hence, a solid basis of in vivo studies with livestock in physiological and productive phases, particularly sensitive to disorders in mineral status and in the gut microbiota, is needed for drawing final conclusions.

Potential of milk mid-infrared spectra to predict nitrogen use efficiency of individual dairy cows in early lactation.

Improving nitrogen use efficiency (NUE) at both the individual cow and the herd level has become a key target in dairy production systems, for both environmental and economic reasons. Cost-effective and large-scale phenotyping methods are required to improve NUE through genetic selection and by feeding and management strategies. The aim of this study was to evaluate the possibility of using mid-infrared (MIR) spectra of milk to predict individual dairy cow NUE during early lactation. Data were collected from 129 Holstein cows, from calving until 50 d in milk, in 3 research herds (Denmark, Ireland, and the UK). In 2 of the herds, diets were designed to challenge cows metabolically, whereas a diet reflecting local management practices was offered in the third herd. Nitrogen intake (kg/d) and nitrogen excreted in milk (kg/d) were calculated daily. Nitrogen use efficiency was calculated as the ratio between nitrogen in milk and nitrogen intake, and expressed as a percentage. Individual daily values for NUE ranged from 9.7 to 81.7%, with an average of 36.9% and standard deviation of 10.4%. Milk MIR spectra were recorded twice weekly and were standardized into a common format to avoid bias between apparatus or sampling periods. Regression models predicting NUE using milk MIR spectra were developed on 1,034 observations using partial least squares or support vector machines regression methods. The models were then evaluated through (1) a cross-validation using 10 subsets, (2) a cow validation excluding 25% of the cows to be used as a validation set, and (3) a diet validation excluding each of the diets one by one to be used as validation sets. The best statistical performances were obtained when using the support vector machines method. Inclusion of milk yield and lactation number as predictors, in combination with the spectra, also improved the calibration. In cross-validation, the best model predicted NUE with a coefficient of determination of cross-validation of 0.74 and a relative error of 14%, which is suitable to discriminate between low- and high-NUE cows. When performing the cow validation, the relative error remained at 14%, and during the diet validation the relative error ranged from 12 to 34%. In the diet validation, the models showed a lack of robustness, demonstrating difficulties in predicting NUE for diets and for samples that were not represented in the calibration data set. Hence, a need exists to integrate more data in the models to cover a maximum of variability regarding breeds, diets, lactation stages, management practices, seasons, MIR instruments, and geographic regions. Although the model needs to be validated and improved for use in routine conditions, these preliminary results showed that it was possible to obtain information on NUE through milk MIR spectra. This could potentially allow large-scale predictions to aid both further genetic and genomic studies, and the development of farm management tools.

Fatty acid profile of phospholipids and sphingomyelin in milk and regulation of sphingomyelin synthesis of mammary glands in cows receiving increasing levels of crushed sunflower seeds.

The objective of this study was to investigate the effect of increasing dietary supplementation of crushed sunflower seed (CSS) in the diet of dairy cows on the fatty acid (FA) composition of phospholipids and sphingomyelin in milk, and on mammary transcription of genes that are important for sphingomyelin de novo synthesis. Four groups of 6 cows received diets supplemented with CSS at 0% (control), or 5, 10, or 15% of dry matter for a 5-wk experimental period. Milk samples and mammary biopsies were collected at the end of the experiment. Phospholipid concentration in milk fat decreased linearly with CSS supplementation. Sphingomyelin concentration in milk fat was unaffected by CSS supplementation. Daily yield of phospholipids decreased linearly with CSS supplementation. Daily yield of sphingomyelin was not significantly affected. The CSS supplementation linearly increased the proportion of monounsaturated FA in milk phospholipids. The major isomer incorporated into phospholipids was C18:1 (n-9 cis), which showed a linear increase with CSS supplementation. The C22:0 proportion in sphingomyelin increased linearly with CSS supplementation and constituted between 15.2 to 25.4% of total FA in sphingomyelin. However, CSS supplementation linearly decreased C23:0 sphingomyelin. Mammary transcription of serine palmitoyl transferase, long chain subunit 1 and subunit 2, the rate-limiting enzymes in ceramide synthesis, showed a linear decrease with increasing CSS supplementation. In conclusion, the data showed that dietary supplementation of CSS linearly increased the proportion of unsaturated FA and monounsaturated FA in milk phospholipids with no effect on phospholipid concentration. In addition, CSS supplementation linearly decreased n-3 polyunsaturated fatty acid proportion in sphingomyelin. The results further showed that mammary transcription of important genes for sphingomyelin de novo synthesis is regulated by lipid supplementation.

Between- and within-herd variation in blood and milk biomarkers in Holstein cows in early lactation.

Both blood- and milk-based biomarkers have been analysed for decades in research settings, although often only in one herd, and without focus on the variation in the biomarkers that are specifically related to herd or diet. Biomarkers can be used to detect physiological imbalance and disease risk and may have a role in precision livestock farming (PLF). For use in PLF, it is important to quantify normal variation in specific biomarkers and the source of this variation. The objective of this study was to estimate the between- and within-herd variation in a number of blood metabolites (ß-hydroxybutyrate (BHB), non-esterified fatty acids, glucose and serum IGF-1), milk metabolites (free glucose, glucose-6-phosphate, urea, isocitrate, BHB and uric acid), milk enzymes (lactate dehydrogenase and N-acetyl-ß-D-glucosaminidase (NAGase)) and composite indicators for metabolic imbalances (Physiological Imbalance-index and energy balance), to help facilitate their adoption within PLF. Blood and milk were sampled from 234 Holstein dairy cows from 6 experimental herds, each in a different European country, and offered a total of 10 different diets. Blood was sampled on 2 occasions at approximately 14 days-in-milk (DIM) and 35 DIM. Milk samples were collected twice weekly (in total 2750 samples) from DIM 1 to 50. Multilevel random regression models were used to estimate the variance components and to calculate the intraclass correlations (ICCs). The ICCs for the milk metabolites, when adjusted for parity and DIM at sampling, demonstrated that between 12% (glucose-6-phosphate) and 46% (urea) of the variation in the metabolites' levels could be associated with the herd-diet combination. Intraclass Correlations related to the herd-diet combination were generally higher for blood metabolites, from 17% (cholesterol) to approximately 46% (BHB and urea). The high ICCs for urea suggest that this biomarker can be used for monitoring on herd level. The low variance within cow for NAGase indicates that few samples would be needed to describe the status and potentially a general reference value could be used. The low ICC for most of the biomarkers and larger within cow variation emphasises that multiple samples would be needed - most likely on the individual cows - for making the biomarkers useful for monitoring. The majority of biomarkers were influenced by parity and DIM which indicate that these should be accounted for if the biomarker should be used for monitoring.

Potential of milk mid-IR spectra to predict metabolic status of cows through blood components and an innovative clustering approach.

Unbalanced metabolic status in the weeks after calving predisposes dairy cows to metabolic and infectious diseases. Blood glucose, IGF-I, non-esterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB) are used as indicators of the metabolic status of cows. This work aims to (1) evaluate the potential of milk mid-IR spectra to predict these blood components individually and (2) to evaluate the possibility of predicting the metabolic status of cows based on the clustering of these blood components. Blood samples were collected from 241 Holstein cows on six experimental farms, at days 14 and 35 after calving. Blood samples were analyzed by reference analysis and metabolic status was defined by k-means clustering (k=3) based on the four blood components. Milk mid-IR analyses were undertaken on different instruments and the spectra were harmonized into a common standardized format. Quantitative models predicting blood components were developed using partial least squares regression and discriminant models aiming to differentiate the metabolic status were developed with partial least squares discriminant analysis. Cross-validations were performed for both quantitative and discriminant models using four subsets randomly constituted. Blood glucose, IGF-I, NEFA and BHB were predicted with respective R 2 of calibration of 0.55, 0.69, 0.49 and 0.77, and R 2 of cross-validation of 0.44, 0.61, 0.39 and 0.70. Although these models were not able to provide precise quantitative values, they allow for screening of individual milk samples for high or low values. The clustering methodology led to the sharing out of the data set into three groups of cows representing healthy, moderately impacted and imbalanced metabolic status. The discriminant models allow to fairly classify the three groups, with a global percentage of correct classification up to 74%. When discriminating the cows with imbalanced metabolic status from cows with healthy and moderately impacted metabolic status, the models were able to distinguish imbalanced group with a global percentage of correct classification up to 92%. The performances were satisfactory considering the variables are not present in milk, and consequently predicted indirectly. This work showed the potential of milk mid-IR analysis to provide new metabolic status indicators based on individual blood components or a combination of these variables into a global status. Models have been developed within a standardized spectral format, and although robustness should preferably be improved with additional data integrating different geographic regions, diets and breeds, they constitute rapid, cost-effective and large-scale tools for management and breeding of dairy cows.

Effect of substituting soybean meal and canola cake with grain-based dried distillers grains with solubles as a protein source on feed intake, milk production, and milk quality in dairy cows.

The growth of the bioethanol industry is leading to an increase in the production of coproducts such as dried distillers grains with solubles (DDGS). Both corn-based DDGS and grain-based DDGS (gDDGS; defined as originating from grain sources such as barley, wheat, triticale, or a mix, excluding corn) appear to be relevant sources of feed and protein for dairy cows. To date, most of the studies investigating DDGS have been performed with corn-based DDGS. The objectives of this study were to determine the effects of the proportion of gDDGS in the diet on feed intake, milk production, and milk quality. The present experiment involved 48 Holstein cows in a replicated 3 × 3 Latin square design with 3 grass-based dietary treatments consisting of 4, 13.5, and 23% gDDGS on a dry matter (DM) basis (L, M, and H, respectively) as a replacement for a concentrate mix. The concentrate mix consisted of soybean meal, canola cake, and beet pulp. Dry matter intake and energy-corrected milk yield were not affected by the proportion of gDDGS in the diet. Daily milk yield decreased with the H diet compared with the L and M diets. The percentage of fat in milk was higher when cows were fed the H diet compared with the L and M diets, whereas milk fat yield was not affected by dietary treatment. The M diet had a higher percentage of protein in milk compared with the L and H diets. Milk protein yield was similar for the L and M diets; however, it decreased for the H diet. Milk taste was not affected by the proportion of gDDGS in the diet or when milk was stored for 7 d. Linoleic acid and conjugated linoleic acid cis-9,trans-11 in milk increased with increasing proportion of gDDGS. To conclude, gDDGS can replace soybean meal and canola cake as a protein source in the diet of dairy cows. Up to 13.5% of the diet may consist of gDDGS without negatively affecting milk production, milk quality, or milk taste. When gDDGS represents 23% of dietary DM, milk production is reduced by 1.6 kg/d, whereas energy-corrected milk production is numerically reduced by 1 kg.

Effect of substituting soybean meal and canola cake with dried distillers grains with solubles at 2 dietary crude protein levels on feed intake, milk production, and milk quality in dairy cows.

Dried distillers grain with solubles (DDGS) is an alternative source of feed protein for dairy cows. Previous studies found that DDGS, based on grains other than corn, can substitute for soybean meal and canola cake as a dietary protein source without reducing milk production or quality. As societal concerns exist, and in many areas strict regulation, regarding nitrogen excretion from dairy cows, the dairy industry has focused on reducing dietary protein level and nitrogen excretion. In the present study, we investigated the use of DDGS as a protein source, at a marginally low dietary crude protein (CP) levels, in a grass-clover and corn silage-based ration. The experiment involved 24 Holstein cows and 2 protein sources (DDGS or soybean-canola mixture) fed at 2 levels of CP (14 or 16%) in a 4 × 4 Latin square design. The aim of this study was to evaluate the effect of both protein source and protein level on feed intake, milk yield, and milk quality. The results indicated that feed intake, milk yield, and protein in milk increased when the protein level in the ration was 16% CP compared with 14%. We found no effect of substituting the soybean-canola mixture with DDGS. Moreover, no sensory problems were observed when comparing fresh milk with stored milk, and milk taste was unaffected by DDGS. Milk from cows fed DDGS had a slightly higher content of linoleic acid and conjugated linoleic acid (CLA 9-11), and lower content of C11 to C17 fatty acids than cows fed diets with the soybean-canola mixture. Cows fed the diets with 16% CP produced milk with higher oleic acids and lower palmitic acid content than cows fed 14% CP diets. To conclude, DDGS can substitute for a soybean-canola mixture without affecting feed intake, milk yield and quality, or sensory quality. Under the conditions of this experiment, feeding 16% CP compared with 14% CP in the ration can increase feed intake and milk production.

Milk yield and estrous behavior during eight consecutive estruses in Holstein cows fed standardized or high energy diets and grouped according to live weight changes in early lactation.

Cows managed for extended lactation go through several estruses before rebreeding. The aims of this study were (1) to quantify the effect of the first 8 estruses after calving on milk yield, milking frequency, and estrous behavioral activity, and (2) to determine the effects of early lactation live weight gain (LWG) as an indication of energy balance on milk yield, plasma insulin-like growth factor 1 (IGF-1) concentration, estrous behavioral activity, interval from calving to first estrus, between-estrus intervals, and pregnancy risk. Milk yield, live weight, and estrous behavioral activity were measured daily in 62 Holstein cows, 17 primiparous and 45 multiparous, managed for an 18-mo calving interval. Blood plasma obtained at wk 3, 5, 12, and 24 after calving was analyzed for IGF-1. Estrus was detected by use of milk progesterone profiles combined with visual observations (i.e., mounting behavior and other). The cows were divided into 2 groups: the cows having a negative LWG in each of the first 5 wk postpartum and the cows having a positive LWG in at least 1 of the first 5 wk after calving. The results indicate a similar decrease of 0.56 kg of milk per day of estrus during each of the 8 consecutive estruses. The activity level was 17 ± 1 movements per hour higher during the 8 estruses compared with the basic activity level. More cows expressed mounting behavior at estrus 8 than at estrus 2 (63.3 and 45.9%, respectively). The negative LWG cows had lower IGF-1 and higher milk production than the positive LWG cows. Both LWG groups had similar interval from calving to first estrus, on average 55 d. To conclude, the decrease in milk yield during estrus is marginal and similar in consecutive estruses. Moreover, estrous behavior is more highly expressed in the later estruses compared with the earlier estruses. Reproductive parameters (frequency of mounting, pregnancy risk, interval to first estrus, and between-estrus intervals) were not influenced by the live weight change during early lactation.

Mechanistic model to predict colostrum intake based on deuterium oxide dilution technique data and impact of gestation and prefarrowing diets on piglet intake and sow yield of colostrum.

The aims of the present study were to quantify colostrum intake (CI) of piglets using the D2O dilution technique, to develop a mechanistic model to predict CI, to compare these data with CI predicted by a previous empirical predictive model developed for bottle-fed piglets, and to study how composition of diets fed to gestating sows affected piglet CI, sow colostrum yield (CY), and colostrum composition. In total, 240 piglets from 40 litters were enriched with D2O. The CI measured by D2O from birth until 24 h after the birth of first-born piglet was on average 443 g (SD 151). Based on measured CI, a mechanistic model to predict CI was developed using piglet characteristics (24-h weight gain [WG; g], BW at birth [BWB; kg], and duration of CI [D; min]: CI, g=-106+2.26 WG+200 BWB+0.111 D-1,414 WG/D+0.0182 WG/BWB (R2=0.944). This model was used to predict the CI for all colostrum suckling piglets within the 40 litters (n=500, mean=437 g, SD=153 g) and was compared with the CI predicted by a previous empirical predictive model (mean=305 g, SD=140 g). The previous empirical model underestimated the CI by 30% compared with that obtained by the new mechanistic model. The sows were fed 1 of 4 gestation diets (n=10 per diet) based on different fiber sources (low fiber [17%] or potato pulp, pectin residue, or sugarbeet pulp [32 to 40%]) from mating until d 108 of gestation. From d 108 of gestation until parturition, sows were fed 1 of 5 prefarrowing diets (n=8 per diet) varying in supplemented fat (3% animal fat, 8% coconut oil, 8% sunflower oil, 8% fish oil, or 4% fish oil+4% octanoic acid). Sows fed diets with pectin residue or sugarbeet pulp during gestation produced colostrum with lower protein, fat, DM, and energy concentrations and higher lactose concentrations, and their piglets had greater CI as compared with sows fed potato pulp or the low-fiber diet (P<0.05), and sows fed pectin residue had a greater CY than potato pulp-fed sows (P<0.05). Prefarrowing diets affected neither CI nor CY, but the prefarrowing diet with coconut oil decreased lactose and increased DM concentrations of colostrum compared with other prefarrowing diets (P<0.05). In conclusion, the new mechanistic predictive model for CI suggests that the previous empirical predictive model underestimates CI of sow-reared piglets by 30%. It was also concluded that nutrition of sows during gestation affected CY and colostrum composition.

Liver protein expression in young pigs in response to a high-fat diet and diet restriction.

We investigated the liver response in young pigs to a high-fat diet (containing 25% animal fat) and diet restriction (equivalent to 60% of maintenance) using differential proteome analysis. The objective was to investigate whether young pigs can be used to model the liver response in adolescents to a high-fat diet and diet restriction-induced BW loss. The high-fat diet increased (P<0.05) the subcutaneous and visceral fat deposition by 45 and 56%, respectively. However, the young pigs on the high-fat diet had normal glucose tolerance and liver lipid content despite a general increase (P<0.05) in plasma lipids (i.e., NEFA, triglycerides, phospholipids, total cholesterol, and lipoproteins). In addition, diet restriction in young pigs induced a modest BW loss (0.7 kg/d; P<0.01) through increased fat mobilization whereas the concentrations of plasma phospholipids, total cholesterol, and low-density lipoprotein decreased (P<0.05) by 37, 36, and 38%, respectively. Data from the proteome analysis indicate that the liver response to a high-fat diet in young pigs is similar to that of humans in terms of increased fatty acid oxidation whereas the liver response to diet restriction is similar to humans in terms of increased gluconeogenesis and glycogenolysis and decreased urea synthesis. Our results suggest that 5 liver proteins, namely acyl-CoA synthetase long-chain 1, sterol carrier protein 2, apolipoprotein C-III, liver fatty acid binding protein, and acyl-CoA-binding protein, play a role in intracellular lipid transport and export in young pigs. In contrast to humans, our results indicate that young pigs are resistant to fat-induced liver lipid accumulation whereas diet restriction decreases fatty acid oxidation and the subsequent ketogenesis in the liver. Consequently, the liver response in adolescents to a high fat diet and diet restriction-induced BW loss cannot reliably be reproduced in young pigs.

Milk production in sows from a teat in second parity is influenced by whether it was suckled in first parity.

The impacts of a teat being suckled or not in first parity on its development, gene expression, and milk yield in the next parity were studied. Forty-seven first-parity sows (Sus scrofa) were divided into 2 groups: i) the same teats suckled in 2 subsequent lactations (controls, CTL; n = 22); and ii) different teats suckled in 2 subsequent lactations (treated, TRT; n = 25). In the first lactation, over half of the teats (Teats 1, 2, 5, 6, and 7 from 1 side of the udder, and Teats 3, 4, and 7 from the other side) were sealed with tape so that they were nonfunctional. During the next lactation, the CTL group had the same teats sealed as in the first lactation, whereas the opposite teats were sealed for the TRT group. In both parities, litters were standardized to 7 piglets around birth and to 6 piglets (1 piglet per available teat) at 48 h postpartum. During the second lactation, piglets were weighed at birth and on d 2, 4, 7, 14, 21, 35, and 56 postpartum. Weaning was at 17 ± 1 d of age. Behavioral measures were obtained (using 24-h video recording) on d 3 and d 10 of lactation on 15 sows per treatment to evaluate satiety of piglets, using aggressiveness and nursing behavior as indicators. At weaning in the second lactation, 16 sows per treatment were slaughtered and 4 functional mammary glands were collected for compositional analyses and parenchyma from 2 nonfunctional glands was collected to measure mRNA abundance for selected genes. Piglets from CTL sows weighed 1.12 kg more than piglets from TRT sows (P < 0.05) on d 56, and functional mammary glands from CTL sows contained more parenchymal tissue, more DNA, and more RNA (P < 0.01) than those from TRT sows. The relative mRNA abundance of prolactin in parenchymal tissue tended to be greater in CTL than TRT sows (P < 0.10). Behavioral measures indicated a greater hunger level for piglets using teats that were not previously suckled. Current findings clearly show that teats that were suckled in first lactation produce more milk and have a greater development in the second lactation than nonsuckled teats.

Liver protein expression in dairy cows with high liver triglycerides in early lactation.

Fatty liver is a frequent subclinical health disorder in dairy cows that may lead to disorders related to the liver function. However, the effect of triglyceride (TG) accumulation on liver metabolic pathways is still unclear. The objective was, therefore, to characterize quantitative differences in the liver proteome between early lactation dairy cows with a low or high liver TG content. The liver proteome analysis indicated that a high liver TG content in early lactation dairy cows is associated with increased oxidation of saturated fatty acids, oxidative stress, and urea synthesis and decreased oxidation of unsaturated fatty acids. Furthermore, liver gluconeogenesis is apparently not impaired by an increased liver TG content. Based on correlations between liver proteins and plasma components, we suggest that future studies investigate the sensitivity and specificity of plasma aspartate aminotransferase, ß-hydroxybutyrate, total bilirubin, total bile acids, and γ-glutamyltransferase for potential use as blood-based biomarkers for early detection of fatty liver in dairy cows. Our study is the first to study the proteome of dairy cows with naturally occurring fatty liver in early lactation.

Effects of nutrient supply, plasma metabolites, and nutritional status of sows during transition on performance in the next lactation.

The aim of the present study was to evaluate the effects of nutrient supply, plasma metabolites, and nutritional status of sows during the transition from gestation to lactation on performance of piglets during the colostral period and throughout lactation. Forty second-parity sows were fed 1 of 4 gestation diets containing a different quantity of dietary fiber (171 to 404 g/kg of DM) from mating until d 108 of gestation. From d 108 of gestation until weaning (d 28 of lactation), sows were fed 1 of 5 lactation diets with a different quantity of dietary fat [3 or 8% with different proportions of medium- (MCFA) and long-chain fatty acids (LCFA)]. Blood was obtained by jugular venipuncture on d 108 and 112 of gestation and on d 1 of lactation, and concentrations of plasma glucose, NEFA, lactate, acetate, propionate, butyrate, and fatty acids were analyzed. Piglet growth and mortality were noted throughout lactation. Piglet mortality during the colostral period (0 to 24 h) was affected by the lactation diets and was positively related to sow backfat (d 108) and plasma lactate (d 112) and negatively related to mean piglet birth weight (P < 0.05). Mean piglet live BW gain (LWG) was recorded in the periods 0 to 24 h, 7 to 10 d, 14 to 17 d, and 17 to 28 d relative to parturition as indirect measures of colostrum yield (0 to 24 h), milk yield in early lactation (d 7 to 10), and at peak lactation (d 14 to 17 and d 17 to 28). Effects of gestation and lactation diets on studied sow traits were tested on selected days during the transition period and the next lactation, and tested statistically on separate days. The LWG in the colostral period was positively correlated with mean piglet birth weight (P < 0.001), plasma concentrations of propionate and MCFA (P < 0.05), and plasma acetate and butyrate (P < 0.1) on d 1 of lactation. The LWG in early lactation was inversely correlated with plasma lactate on d 108 (P < 0.05), plasma glucose on d 112, and backfat thickness on d 108 (P < 0.10). The LWG at peak lactation was positively correlated with MCFA intake of the sow on d 113 to 115 and backfat thickness on d 108 during the transition, and negatively correlated with intake of LCFA and ME intake on d 108 to 112 (P < 0.05). In conclusion, feeding and body condition of sows during the transition from gestation to lactation is important for neonatal piglet survival, lactation performance of sows, and piglet growth during the next lactation.

Effects of gestation and transition diets, piglet birth weight, and fasting time on depletion of glycogen pools in liver and 3 muscles of newborn piglets.

The experiment was conducted to assess the effects of maternal nutrition in late gestation on glycogen pools of newborn piglets of different birth weights and to assess how rapidly the glycogen pools in the liver and 3 muscles are mobilized during fasting. Until d 108 of gestation, 48 sows were fed a gestation standard diet (GSD) with low dietary fiber (DF, 17.1%), or 1 of 3 diets with high DF (32.3 to 40.4%) consisting of pectin residue (GPR), potato pulp (GPP), or sugar-beet pulp (GSP). From d 108 until farrowing, sows were fed 1 of 6 transition diets with low or high dietary fat: one group received a standard diet (TSD; control) containing 3% animal fat, another group received the TSD diet + 2.5 g/d of hydroxy-methyl butyrate as topdressing (THB), and 4 other groups received diets with 8% added fat from coconut oil (TCO), sunflower oil (TSO), fish oil (TFO), or 4% octanoic acid + 4% fish oil (TOA). Two piglets per litter (the second and fifth born) were blood sampled, and 1 was killed immediately after birth, whereas the other, depending on the litter, was killed after 12, 24, or 28.5 to 36 h (mean 32.5 h) of fasting. Samples of liver, LM, M. semimembranousus (SM), and M. diaphragm (DP) were collected and analyzed for glycogen concentration. No dietary effects (P > 0.20) on glycogen concentrations in liver, LM, SM, or DP were observed. The weight of the liver was affected by gestation diet (P < 0.05) and was greater in GSD and GSP piglets (36.7 and 36.3 g) than in GPR piglets (32.6 g), and intermediate (33.6 g) in GPP piglets. Liver weight, estimated muscle mass, and glycogen pools (P < 0.001) were affected by birth weight, whereas glycogen concentrations in liver and LM, SM, and DP muscles were not (P > 0.05). Liver weight; glycogen concentrations in liver, LM, SM, and DP; and glycogen pools in liver and muscles decreased (P < 0.001) with increasing duration of fasting, and at 32.5 h of fasting, glycogen concentration was reduced by 80% in liver, 64% in DP, 46% in SM, and 36% in LM. Based on a broken-line model, labile glycogen in SM, a locomotory muscle, was estimated to be depleted after 16.4 h of fasting. In conclusion, piglet size had a major impact on estimated glycogen pools, whereas sow nutrition in late gestation had a minor impact, if any. Furthermore, varying proportions of pools of glycogen present in liver and selected muscles were mobilized, and data indicate that newborn piglets are fatally depleted of energy after 16 h of fasting.

Identification of ABCA1 and ABCG1 in milk fat globules and mammary cells--implications for milk cholesterol secretion.

The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play an important role in cellular cholesterol homeostasis, but their function in mammary gland (MG) tissue remains elusive. A bovine MG model that allows repeated MG sampling in identical animals at different functional stages was used to test whether 1) ABCA1 and ABCG1 protein expression and subcellular localization in mammary epithelial cells (MEC) change during the pregnancy-lactation cycle, and 2) these 2 proteins were present in milk fat globules (MFG). Expression and localization in MEC were investigated in bovine MG tissues at the end of lactation, during the dry period (DP), and early lactation using immunohistochemical and immunofluorescence approaches. The presence of ABCA1 and ABCG1 in MFG isolated from fresh milk was determined by immunofluorescence. The ABCA1 protein expression in MEC, expressed as arbitrary units, was higher during the end of lactation (12.2±0.24) and the DP (12.5±0.22) as compared with during early lactation (10.2±0.65). In contrast, no significant change in ABCG1 expression existed between the stages. Throughout the cycle, ABCA1 and ABCG1 were detected in the apical (41.9±24.8 and 49.0±4.96% of cows, respectively), basal (56.2±28.1 and 54.6±7.78% of cows, respectively), or entire cytoplasm (56.8±13.4 and 61.6±14.4% of cows, respectively) of MEC, or showed combined localization. Unlike ABCG1, ABCA1 was absent at the apical aspect of MEC during early lactation. Immunolabeling experiments revealed the presence of ABCA1 and ABCG1 in MFG membranes. Findings suggest a differential, functional stage-dependent role of ABCA1 and ABCG1 in cholesterol homeostasis of the MG epithelium. The presence of ABCA1 and ABCG1 in MFG membranes suggests that these proteins are involved in cholesterol exchange between MEC and alveolar milk.

Differential expression and localization of lipid transporters in the bovine mammary gland during the pregnancy-lactation cycle.

The transport of lipids across mammary gland epithelial cells (MEC) determines milk lipid content and composition. We investigated the expression of lipid transporters and their regulators in comparison to blood metabolites during lactation and dry period (DP) in dairy cows. Repeated mammary gland biopsies and blood samples were taken from 10 animals at 7 stages of the pregnancy-lactation cycle. Expression levels of the specific mRNAs were determined by quantitative reverse transcription-PCR, whereas ABCA1 was localized by immunohistochemistry. Blood serum metabolites were determined by common enzymatic chemistries. Elevated mRNA profiles of ABCA1 and ABCA7 were found during DP as compared with lactation and were inversely associated with blood cholesterol levels. Elevated levels of ABCG2, NPC1, SREBP1, SREBP2, LXR alpha, and PPAR gamma were found postpartum, whereas ABCG1 did not differ between the functional stages of the mammary gland. The ABCA1 protein was localized in MEC and showed differential activity between DP and lactation suggesting a role of ABCA1 in the removal of excess cellular cholesterol from MEC during the DP. The expression profiles of ABCA7 and NPC1 may reflect a role of these transporters in the clearance of apoptotic cells and the intracellular redistribution of cholesterol, respectively. Regulation of lipid transporters in the mammary gland is partially associated with transcription factors that control lipid homeostasis.

No effect of the plant growth regulator, chlormequat, on boar fertility.

Chlormequat is a commonly used plant growth regulator in agriculture. Defined levels of chlormequat residue are allowed in food and an acceptable daily intake is defined for humans. However, there are results in the literature suggesting that a daily intake below the acceptable level for human is detrimental for mammalian reproduction. In the present experiment we investigated the effect of chlormequat at levels up to that acceptable for humans on reproduction in male pigs. Chlormequat (also known as chlorocholine chloride (CCC)) was mixed into the diet and given to the experimental animals at three levels (three treatment groups), i.e. 0 mg CCC/kg BW per day (Control), 0.025 mg CCC/kg BW per day and 0.05 mg CCC/kg BW per day. Eight mother sows per treatment group were used in the experiment. From the day of insemination, the mother sows received the experimental diets. The piglets were weaned at 4 weeks of age and two boar littermates continued on the same treatment as the dam until maturity and delivery of semen for in vitro fertilization (IVF) and in vivo fertilization. Semen volume, sperm concentration and fraction of live sperms were not (P 0.46) detrimentally affected by chlormequat intake. The fraction of oocytes developing to more than the one-cell stage at day 5 after IVF was not (P = 0.88) detrimentally affected by chlormequat intake. Chlormequat intake did not detrimentally affect the fraction of gilts being pregnant after one insemination (P = 0.65) or the number of embryos in the pregnant gilts (P = 0.36). Serum chlormequat concentration was 0.9 µg/kg in the 0.025 mg CCC/kg BW per day group and 1.8 µg/kg in the 0.05 mg CCC/kg BW per day group, but was below the detection limit in control animals. In conclusion, the plant growth regulator chlormequat could not be proven to be detrimental to the selected reproduction traits in male pigs. This is in contrast to existing results from the male mouse.

Cellular mechanisms in regulating mammary cell turnover during lactation and dry period in dairy cows.

The mechanisms involved in regulating mammary cell turnover during the pregnancy-lactation cycle in dairy cows are unclear. The objective of present experiment was to describe expression of genes encoding proteins known to be involved in pathways regulating mammary cell proliferation, apoptosis, differentiation, cell survival, and tissue remodeling. Mammary gland biopsies were taken 7 times during the pregnancy-lactation cycle of 10 dairy cows, and samples were analyzed by immunohistochemistry and real-time PCR. Cell proliferation was greatest during the dry period and apoptosis was high in early dry period and early lactation. Based on Fas (tumor necrosis factor receptor superfamily member 6), Fas ligand, and caspase-3, caspase-8, and caspase-9 gene expression, no indication was found of a stage-dependent shift between the extrinsic and intrinsic pathways leading to apoptosis. Gene expression of microsomal glutathione S-transferase (mGST) did not vary significantly, whereas B-cell leukemia/lymphoma 2 (Bcl-2) and BCL2-associated X protein (Bax) gene expression was greatest during the dry period and early lactation and coincided with high cell turnover. Gene expression of early response genes c-Fos, c-Jun, and c-Myc correlated to neither rate of cell proliferation nor plasma concentration of insulin-like growth factor (IGF)-I and insulin. Gene expression of nuclear factor of kappa light chain gene enhancer in B-cells (NFkappaB) and NFkappaB inhibitor alpha was greatest in the periparturient period, and NFkappaB gene expression coincided with an anticipated need for cell survival factors. Expression of transforming growth factor beta (TGF-beta) receptor 1 and 2 mRNA was greatest in early lactation, whereas TGF-beta1 did not vary significant during the pregnancy-lactation cycle. Even though our results on the TGF-beta system did not comply with other studies, the gene expression pattern of the TGF-beta receptors indicates a role in regulating apoptosis in early lactation. Signal transducer and activator of transcription 5 (STAT5) gene expression was high in the periparturient period, which suggests a role for STAT5 in regulation of mammary cell proliferation and differentiation in dairy cows. Expression of tissue-plasminogen activator, plasminogen activator inhibitor-1, and IGF binding protein 5 genes was greatest in early lactation, suggesting a role for IGF binding protein 5 in coordinating regulation of apoptosis and tissue remodeling.

Effect of pregnancy and feeding level on cell turnover and expression of related genes in the mammary tissue of lactating dairy cows.

Milk yield is reduced by pregnancy, and the present experiment was conducted to study the biological basis for the negative effect of pregnancy on milk yield. A total of 16 dairy cows were fed at either a normal or a low feeding level (eight cows per treatment), and half of them were inseminated after approximately 3 months of lactation and the other half were not inseminated. Mammary biopsies were taken at approximately 9 months of lactation. The milk yield of pregnant cows was reduced by 2.6 kg/day, and lactation persistency was reduced already from the time of insemination. Low feeding level reduced the milk yield by 9.8 kg/day from week 8 to week 39 of lactation, whereas no interaction between pregnancy and feeding level was found. Cell proliferation (Ki-67) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL) were unaffected by feeding level, and pregnancy tended to reduce cell proliferation but had no effect on apoptosis. Reduced cell proliferation may explain the reduced lactation persistency in pregnant cows. Transcription of oestrogen receptor α, progesterone receptor A and B, and long and short isoforms of the prolactin receptor were higher in pregnant cows compared with non-pregnant cows. Feeding level did not mediate changes in transcription of genes. Transcription of other cell-turnover-related genes (IGF-I, IGF binding protein-5, caspase-3) as well as genes related to the secretory activity of the cells (α-lactalbumin and acetyl CoA carboxylase α) was not affected by pregnancy or by feeding level.

Determination of chlormequat in pig serum and sow milk by LC-MS/MS.

Chlormequat is a plant growth regulator widely used on cereals, and there is general concern that it may impair human fertility. A LC-MS/MS method for the analysis of chlormequat in milk and serum was developed and validated in connection with an investigation on the effect of chlormequat on pig reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol-water-acetic acid, centrifuged, filtrated and determined by LC-MS/MS. The mean recoveries were in the range 80-110%, and the LOD was 0.2 ng/g for serum and 0.3 ng/g for milk. The values for repeatability and reproducibility were within 2/3 of the limits given by the Horwitz equation. Samples of pig serum (59) and sow milk (27) were analyzed using the method. Chlormequat was determined in four milk samples in the range of 0.4 ng/g to 1.2 ng/g and in all serum samples in the range of 0.2 ng/g-4.0 ng/g.