Dry off accelerates apoptosis and involution of the mammary gland (Capuco and Akers, Reference Capuco and Akers1999; Caja et al., Reference Caja, Salama and Such2006; Zhao et al., Reference Zhao, Ponchon, Lanctôt and Lacasse2019). Feed restriction may be used to decrease milk synthesis at the end of lactation in cows (Herve et al., Reference Herve, Quesnel, Veron, Portanguen, Gross, Bruckmaier and Boutinaud2019; Vanacker et al., Reference Vanacker, Girard, Blouin and Lacasse2020) and improve the efficiency of mammary involution after milking cessation (Ollier et al., Reference Ollier, Zhao and Lacasse2014). Dairy goats are usually managed in the same way, although they have rather persistent lactations, and some may continue to produce milk through until the next parturition if milking continues (Salama et al., Reference Salama, Caja, Such, Casals and Albanell2005; Caja et al., Reference Caja, Salama and Such2006; Safayi et al., Reference Safayi, Theil, Hou, Engbaek, Nørgaard, Sejrsen and Nielsen2010). Stress and glucocorticoids release have been associated with increased apoptosis and decreased milk production in cows and sheep (Caroprese et al., Reference Caroprese, Albenzio, Marzano, Schena, Annicchiarico and Sevi2010; Ollier et al., Reference Ollier, Beaudoin, Vanacker and Lacasse2016; Ponchon et al., Reference Ponchon, Zhao, Ollier and Lacasse2017), however, whether this might also affect apoptosis and involution during the dry period of goats remains unclear.
Feed restriction and then abrupt cessation of milking are considered stressful management (Odensten et al., Reference Odensten, Holtenius and Waller2007; Zobel et al., Reference Zobel, Leslie, Weary and Von Keyserlingk2013). Some authors have also associated milk accumulation during the dry period to stress, inflammation of the mammary gland and a higher risk of mastitis (Bertulat et al., Reference Bertulat, Fischer-Tenhagen, Suthar, Möstl, Isaka and Heuwieser2013; Mehdid et al., Reference Mehdid, Martí-De Olives, Fernández, Rodríguez and Peris2019; Zhao et al., Reference Zhao, Ponchon, Lanctôt and Lacasse2019). Other studies have reported increased infiltration of immune cells and an increased rate of apoptosis in mammary tissue within a few hours or days after the start of the dry off (Boutinaud et al., Reference Boutinaud, Isaka, Lollivier, Dessauge, Gandemer, Lamberton, Taranilla, Deflandre and Sordillo2016; Lanctôt et al., Reference Lanctôt, Fustier, Taherian, Bisakowski, Zhao and Lacasse2017; Singh et al., Reference Singh, Phyn, Reinsch, Dobson, Oden, Davis, Stelwagen, Henderson and Molenaar2017). Moreover, milking cessation is marked by decreased casein and lactalbumin synthesis and an increase of lactoferrin synthesis in mammary tissue (Singh et al., Reference Singh, Phyn, Reinsch, Dobson, Oden, Davis, Stelwagen, Henderson and Molenaar2017). In addition, the reduction of alveolar tissue can be observed within weeks after the start of dry off (Capuco et al., Reference Capuco, Wood, Baldwin, Mcleod and Paape2001; Singh et al., Reference Singh, Phyn, Reinsch, Dobson, Oden, Davis, Stelwagen, Henderson and Molenaar2017).
The dry off coincides with the estrogen changes that characterize the acceleration of mammary involution and the end of pregnancy. These changes induce the renewal of epithelial cells, resulting in a lobe-alveolar structure that ensures copious milk synthesis during the next lactation (Safayi et al., Reference Safayi, Theil, Hou, Engbaek, Nørgaard, Sejrsen and Nielsen2010; Zhao et al., Reference Zhao, Ponchon, Lanctôt and Lacasse2019). In this context, the analyses of the expression of casein (CSN2), lactalbumin (LALBA), and lactoferrin (LF) genes related to capacity synthesis and immunomodulation in mammary tissue during dry off are essential to understand the involution (Singh et al., Reference Singh, Dobson, Phyn, Davis, Farr, Molenaar and Stelwagen2005, Reference Singh, Phyn, Reinsch, Dobson, Oden, Davis, Stelwagen, Henderson and Molenaar2017). Likewise, the expression of insulin-like growth factor 1 receptor (IGF1R) genes and phosphatidylinositol-3-kinase (PIK3CA), protein kinase B (AKT1), mechanistic target of rapamycin (MTOR), all of which are related to apoptosis control in mammary epithelial cell line (Burgos et al., Reference Burgos and Cant2010; Li et al., Reference Li, Liu, Wang, Lin, Yan, Cao, Zhao and Shi2017), can contribute to understanding the effects of stress and cortisol release during dry off.
We hypothesized that stress induced experimentally by ACTH administration and cortisol release would increase SCC in mammary secretions, and improve the effectiveness of dry off in Saanen goats. Our objective was to evaluate the effect of ACTH administration on the expression of genes related to milk synthesis (LALBA, CSN2) and antimicrobial action (LF), genes related to cell survival and apoptosis control (IGF1R, PIK3CA, AKT1, MTOR) in mammary tissue, and its relationship with lactoferrin concentration, SCC and leukocyte count in mammary secretions of Saanen goats during dry off.
Materials and methods
Experimental procedures were approved by the Animal Ethics Committee (protocol 3709280316) of the Faculty of Animal Science and Food Engineering in accordance with Brazilian federal law.
Housing, food, and management
The Saanen goats remained in collective pens with free access to feed and water troughs and mineral salt. The goats were fed with 50% roughage (containing corn silage) and 50% concentrate (corn and soybean meal, soybean oil, limestone and mineral and vitamin mix). During lactation, the diet was adjusted every 2 weeks, considering the live weight and milk production to ensure leftovers of 15% of the total diet provided (NRC, 2007). Throughout lactation, the experimental goats were subjected to one daily milking (7 am). In the last milking, all goats received an intramammary antibiotic (Ouro Fino, Cravinhos, SP, Brazil) as a preventive measure against mammary gland infections. During the last 3 d of lactation (on day 247 ± 5 of lactation) and after abrupt cessation of milking on day 250, all the goats began receiving a dry off goat diet. This diet contained total restriction of corn silage and concentrate, and the goats received Tifton 85 hay ad libitum for a period of 15 d. Afterwards, the animals received a dry goat diet containing corn silage and concentrate (NRC, 2007). The experimental procedures were performed in 12 h of daylight and 12 h of darkness.
Organization of the experiment
No goats were pregnant during our experiment because the objective was to study the effect of stress, via ACTH administration and cortisol release on mammary involution during dry off without the influence of pregnancy. On day 250 of lactation (experiment day 0), the 30 goats (67.1 ± 2.5 kg of BW, 3.2 ± 0.1 of body score, 2.2 ± 0.5 lactations) were subjected to abrupt dry off and were blocked (according to parity number, number of kids born, total milk yield in current lactation, body weight and body condition score) in two treatments (1) challenge with ACTH (intravenous administration of 0.6 IU/kg per BW per animal) and (2) Control (saline administration).
The ACTH and Control treatments were administered by intravenous injections at 7 am on days 1, 3, 6, 9, 12, 15, 30, and 60 of dry off. The ACTH dose used causes a cortisol peak at 60 min and a return to baseline cortisol concentrations at 240 min after ACTH administration (Fulkerson and Jamieson, Reference Fulkerson and Jamieson1982; Bomfim et al., Reference Bomfim, Merighe, de Oliveira and Negrão2018).
Sampling and analyses
Milk yield (kg/d) was recorded during the last 15 d of lactation. In order, blood samples, milk samples and mammary secretions were collected at 8 am (1 h after ACTH or Control administration) before (day −1, on day 249 of lactation) and after milking cessation of the experimental goats, on days 1, 3, 6, 9, 12, 15, 30, and 60 of the dry period. Milk and mammary secretion samples were taken from both teats manually and mixed about 50 ml to determine the composition, microbiological content, SCC, leukocyte count and lactoferrin concentration. Blood samples were taken by a jugular venepuncture. Cortisol in plasma was determined by an EIA kit (Monobind, Lake Forest, CA, USA), the intra- and inter-assay coefficients of variation were less than 4.3 and 6.8%, respectively. The lactoferrin concentration in milk and mammary secretions was determined by an EIA kit (Bethyl Laboratories, Montgomery, Texas, USA). The fat, protein and lactose levels were determined by an ultrasound equipment (Scope Electric®, Razgrad, Bulgaria). The SCC and leukocyte count in milk and mammary secretions were performed using the direct method (Dulin et al., Reference Dulin, Paape and Wergin1982; Gonzalo et al., Reference Gonzalo, Boixo, Carriedo and San Primitivo2004).
Experimental goats were considered dry when the volume of mammary secretion collected was less than 50 ml (Fleet et al., Reference Fleet, Goode, Hamon, Laurie, Linzell and Peaker1975). Photographs of the udders were assessed on days 1, 3, 6, 9, 12, 15, 30, and 60 of the dry off to visually confirm the involution of the mammary gland (Fowler et al., Reference Fowler, Knight and Foster1991). The involution was considered total when the udder had a morphology similar to those observed in non-lactating and non-pregnant goats.
Gene expression
On days 1, 15, 30, and 60 after the start of dry off, biopsies were performed on the mammary gland of eight goats (four ACTH goats and four Control goats) distributed to milk yield and representative of other experimental goats. Biopsies were also performed 60 min after ACTH or Control administration, as previously described (Bomfim et al., Reference Bomfim, Merighe, de Oliveira and Negrão2018; Hooper et al., Reference Hooper, Silva, de Oliveira, Meringhe, Lacasse and Negrão2020). Briefly, the disposable biopsy needle was inserted into the incision to a depth of approximately 3.0 cm, and the equipment was triggered to cut and collect a sample of approximately 2.0 × 0.5 cm of mammary tissue. The relative levels of gene expression of IGF1R, PIK3CA, AKT1, MTOR, CSN2, LALBA, and LF proteins were determined through real-time quantitative PCR using the StepOne Real-Time PCR System (Invitrogen) as well as the primers described in online Supplementary Table S1. The total RNA of mammary tissue (80 to 100 mg) was extracted and purified using a PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA, USA). The material was treated with RNase-free DNase (Promega, Madison, WI, USA) to prevent genomic DNA contamination. The RNA concentrations were determined using Qubit 2.0 Fluorometric Quantification (Thermo Fisher Scientific, Waltham, MA). The quality of each RNA sample was evaluated by the optical density at the 260 and 280 nm absorbance waves and the 260/280 absorption ratio was approximately 2. The integrity of the RNA was analyzed on denaturing agarose gel. The relative expression of the gene was quantified using the Livak method (ΔΔCt = 2−ΔΔCt) (Livak et al., Reference Livak and Schmittgen2001) to compare the expression level of the target genes with the reference (GAPDH). For each primer set, the PCR efficiency was close to 100%, and the specificity of the primer pairs was confirmed through analysis of the melting curve and appropriate size, using 1.5% agarose gel electrophoresis.
Statistical analysis
Data normality was confirmed using the Shapiro–Wilk test. The effects of treatment (ACTH or Control) on gene expression, cortisol in plasma, lactoferrin concentration, SCC, leukocyte count, and microbiological analysis of mammary secretions during the dry period was analyzed through ANOVA using the MIXED procedure of SAS 9.4, which separated the effects of treatment, day, and goats as causes of variation in the model; treatment and day was considered a fixed effect and goats were considered random. Several covariance matrices were tested, and the one that had the best structure according to Bayesian information and criteria was selected. When there was a significant effect, the means were compared using Fisher's test with a significance level of P ≤ 0.05. Data are presented as mean ± standard error.
Results
As expected, feed restriction significantly decreased the milk yield of the experimental goats (online Supplementary Fig. S1). The cortisol concentration of goats submitted to ACTH was significantly higher than that measured for goats Control (online Supplementary Fig. S2). In addition, the increase in cortisol concentration in Control goats during the dry off confirmed that this practice was stressful for goats (online Supplementary Fig. S2). The results obtained demonstrated that the stress caused via ACTH administration causes a significant increase in the percentage of dry goats at days 6, 15, 30 and 60 compared to the Control (Table 1). At the same time, dry goats from both experimental groups visually presented total udder involution, showing a similar morphology of non-lactating and non-pregnant goats (online Supplementary Fig. S3).
Table 1. Effect of ACTH or Control treatment on percentage of dry goats during the dry period of Saanen goats
1 Experimental goats were considered dry when the volume of mammary secretion collected was less than 50 ml.
2 The percentages were compared using a χ2 test.
a,bPercentage within a row with different superscripts differ (P ≤ 0.05).
The highest cortisol in ACTH goats significantly increased the concentration of lactoferrin, milk density, SCC and percentage of eosinophils and basophils, but decreased the concentration of lymphocytes compared with the mammary secretions of the Control goats (Table 2; online Supplementary Figs S4 and S5). Comparing the treatment effect on gene expression during the entire dry period (calculated as gene expression on day 60 vs. day 1 of dry period for ACTH or Control treatments), ACTH goats significantly downregulated the expression of IGF-1R and upregulated the expression of PIK3CA in mammary tissue when compared with goats submitted to the Control (Fig. 1). Comparing the effect of day biopsy, the expression of AKT1, LF, LALBA, and CSN2 genes on day 1 was significantly higher when compared to day 60 of dry off (Fig.2).
Fig. 1. Downregulation and upregulation of target genes in mammary gland for goats subjected to ACTH and Control during the entire dry period. Gene expression is reported as fold change (2−ΔΔCt) relative to Control. Gene expression during the entire period was presented as gene expression on day 60 vs. day 1 of the dry period for ACTH and Control treatments. The asterisk (*) indicates significant differences (P ≤ 0.05) between ACTH (black bars: gene expression on day 60 vs. day 1) and Control (gray bars: gene expression on day 60 vs. day 1) treatments.
Fig. 2. Gene expression in mammary tissue of experimental goats on days 1 and 60 of dry off. Gene expression is reported as fold change (2−ΔΔCt) relative to Control. The asterisk (*) indicates significant differences (P ≤ 0.05) between day 1 (black bars) and 60 (gray bars) of dry off.
Table 2. Effect of ACTH or Control treatment (T), sampling day (D), and their interaction (T*D) on composition, somatic cell count, leukocyte count, and microbiological analysis in the mammary secretion of Saanen goats during the dry period. Data are presented as the mean ± standard error of the mean.
1 Four goats from ACTH and eight goats from the Control group showed a considerable volume of mammary secretion and were no considered dry goats.
2 Values × 103/ml.
3 CFU: colony-forming units, values × 103/ml.
4 The count was not high enough to perform statistical analysis.
a,bMeans within a row with different superscripts differ (P ≤ 0.05).
Discussion
Feed restriction significantly reduced milk yield before the abrupt cessation of milking as the lower availability of nutrients decreased the milk synthesis by the mammary gland (Ollier et al., Reference Ollier, Beaudoin, Vanacker and Lacasse2016; Herve et al., Reference Herve, Quesnel, Veron, Portanguen, Gross, Bruckmaier and Boutinaud2019; Vanacker et al., Reference Vanacker, Girard, Blouin and Lacasse2020). ACTH administration increased the percentage of dry goats compared to the Control (73 vs. 46%). On the other hand, 56% of goats submitted to Control had persistent lactation just 60 d after the start of dry off. These results confirm that typical dry off management was ineffective, and many goats have a considerable volume of mammary secretion in the mammary gland. Some authors have suggested that feed restriction causes hunger and interruption of milking that causes discomfort and even pain, stressors that increase cortisol concentration during dry off (Bertulat et al., Reference Bertulat, Fischer-Tenhagen, Suthar, Möstl, Isaka and Heuwieser2013; Zobel et al., Reference Zobel, Weary, Leslie and Von Keyserlingk2015). In addition, ACTH administration significantly increased the concentration of cortisol and the percentage of dry goats, improving the efficiency of dry off when compared to Control goats.
Other authors have also reported that abrupt cessation of milking causes milk accumulation, decreases the synthesis of the main constituents of milk and increases the permeability of alveoli, changing the composition of mammary secretions at the start of dry off (Singh et al., Reference Singh, Vetharaniam, Dobson, Prewitz, Oden, Murney, Swanson, McDonald, Henderson and Stelwagen2016, Reference Singh, Phyn, Reinsch, Dobson, Oden, Davis, Stelwagen, Henderson and Molenaar2017). In our study, the higher density and lactoferrin concentration in mammary secretions of goats submitted to ACTH confirms its effect in the mammary gland. The large percentage variability of the main constituents in mammary secretions observed in our study suggested that the dry off effect on goats with persistent lactations is quite heterogeneous. In contrast to that reported by other authors (Fowler et al., Reference Fowler, Knight and Foster1991), our results also demonstrate that dry goats showed an udder morphology similar to that observed in non-pregnant and non-lactating goats. Indeed, goats with persistent lactation during the dry off process also showed visual changes in udder morphology, even when they maintained a considerable volume of secretion in the udder at the end of our study. In our study, goats were not pregnant, and we cannot associate lactation persistency to hormonal variations that characterize the end of pregnancy and accelerate mammary gland renewal, as reported by other authors (Salama et al., Reference Salama, Caja, Such, Casals and Albanell2005; Safayi et al., Reference Safayi, Theil, Hou, Engbaek, Nørgaard, Sejrsen and Nielsen2010).
Furthermore, our results demonstrate that ACTH administration significantly downregulated the expression of IGF1R and upregulated the expression of PIK3CA in mammary tissue compared to the Control. Consequently, as we initially postulated, the increased cortisol levels in ACTH goats changed the expression of genes IGF1R and PIK3CA in the IGF1R/PIK3CA/AKT1/MTOR pathway. However, ACTH administration decreased the expression of IGF1R and increased the expression of PIK3CA, both associated with an anti-apoptotic role and cell survival (Burgos et al., Reference Burgos and Cant2010; Zhang et al., Reference Zhang, Huang, Yi, Zhang, Loor, Cao, Shi and Luo2018). The ACTH effect was higher on the expression of IGF1R than on the expression of PIK3CA, suggesting that this misbalance accelerated mammary involution in goats subjected to ACTH administration. The lower expression of the IGF1R gene in mammary tissue of ACTH goats was also related to an increase in SCC in mammary secretion and a higher percentage of dry goats when compared to Control goats (73.3 vs. 46.7%, respectively). Moreover, ACTH also increased the lactoferrin concentration in mammary secretions when compared to Control. Other authors have reported that in the absence of sub-clinical or clinical mastitis, the increase in SCC is a consequence of the increase in epithelial cells exfoliated in milk (Boutinaud et al., Reference Boutinaud, Isaka, Lollivier, Dessauge, Gandemer, Lamberton, Taranilla, Deflandre and Sordillo2016; Herve et al., Reference Herve, Quesnel, Lollivier and Boutinaud2016). In our study, there was no effect of ACTH on microbiological status or intramammary infections, so we can argue that there was a higher exfoliation of the mammary epithelial cells during the mammary involution of the goats subjected to ACTH administration. Although the total number of mammary epithelial cells decreases during lactation of the goats (Knight and Peaker, Reference Knight and Peaker1984; Safayi et al., Reference Safayi, Theil, Hou, Engbaek, Nørgaard, Sejrsen and Nielsen2010), some authors have argued that the goats have more persistent lactations because their epithelial cells that survive maintain their high capacity for milk synthesis (Knight and Peaker, Reference Knight and Peaker1984; Wilde et al., Reference Wilde, Addey, Li and Fernig1997; Safayi et al., Reference Safayi, Theil, Hou, Engbaek, Nørgaard, Sejrsen and Nielsen2010). These last aspects can partially explain why 54% of goats submitted to the Control and 27% of goats submitted to ACTH had a considerable secretion volume in their mammary gland 60 d after the start of dry off. As previous studies concerning cortisol supplementation in epithelial mammary cells of goats demonstrated increase apoptosis (Bomfim et al., Reference Bomfim, Merighe, de Oliveira and Negrão2018), further studies with ACTH administration are necessary to determine its effects on apoptosis of epithelial cells and mammary involution during the dry period.
In our study, ACTH goats presented significantly higher basophil and eosinophil percentages and lower lymphocyte percentage in mammary secretions than Control goats. This changeover can be attributed to ACTH and cortisol because both are immunosuppressants (Sordillo et al., Reference Sordillo, Shafer-Weaver and DeRosa1997; Gonçalves et al., Reference Gonçalves, Lyman, Hockett, Rodriguez, Dos Santos and Anderson2017; Mehdid et al., Reference Mehdid, Martí-De Olives, Fernández, Rodríguez and Peris2019). In contrast, the neutrophil percentage, the most abundant immune cells during the inflammatory process of mammary tissue in ruminants (Sordillo et al., Reference Sordillo, Shafer-Weaver and DeRosa1997; Gonçalves et al., Reference Gonçalves, Lyman, Hockett, Rodriguez, Dos Santos and Anderson2017), was not influenced by ACTH. For this reason, we argue that the physiological ACTH dose used had a limited effect on the immune response. Furthermore, the ACTH goats had an increased lactoferrin concentration in mammary secretions compared to Control, which might partially explain why our goats did not present clinical mastitis during dry off. Lactoferrin is bacteriostatic and can prevent growth of bacteria such as staphylococci and coliforms, which have iron requirements (Sordillo et al., Reference Sordillo, Shafer-Weaver and DeRosa1997). In addition, administering intramammary antibiotics for all experimental goats was effective, since no goats presented clinical mastitis during dry off. On the other hand, the decrease of lymphocytes in mammary secretions, which actively participate in the immune response in the mammary gland, is a concern and may hinder the use of higher doses of ACTH than that used in our study.
In conclusion, our study shows that ACTH administration significantly downregulated the expression of IGF1R and upregulated the expression of PIK3CA in mammary tissue and increased the lactoferrin concentration and SCC in mammary secretion compared to the Control. ACTH administration significantly increased the percentage of dry goats compared to the Control (73 vs. 46%), therefore, we can conclude that stress induced experimentally by ACTH administration and cortisol release accelerated mammary involution in ACTH goats during the early dry-off.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029922000735.
Acknowledgments
This work was financed by FAPESP (grants 2018/09093 5); This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. J. A. Negrão was supported by the CNPq.
