Effects of breed and milking frequency on udder histological structures in dairy goats

Abstract

Tissue percentages (secretory, connective, ductal and vascular tissues) and the number and size of the alveoli in the udders of three dairy goat breeds under two milking frequencies (once- vs. twice-daily milking) were studied. Nine dairy goats, three of each breed studied (Majorera, Tinerfeña and Palmera), were milked during 6 weeks beginning at the tenth week of lactation. The right udder half was milked twice daily, and the left udder half was milked once daily. Moreover, during the experimental period, morphological udder data, milk yield, milk fractioning and milk composition were recorded. The goats were sacrificed and two samples for each gland were taken for the histological study. The statistical analyses revealed that the histological parameters were not influenced by the milking frequency, and that the breed determines different percentage of tissue components. Correlations between morphological parameters of the udder and milk-yielding parameters were high and determined the greater importance of the globosity and the structure of the udder in the milk production. Furthermore, it was determined that the percentage of secretory tissue in the mammary parenchyma has no correlation with the milk yield parameters in different high-production dairy breeds. Histological parameters (secretory and connective tissues) only have an impact in the milk fractions.

Keywords:

• dairy goat
• udder
• histological parameter
• milking frequency
• tissue

1. Introduction

Morphological characteristics of the tissue components of the udder, in particular correct values of secretor and connective tissues, are important items that deserve more attention in a healthy mammary parenchyma. The secretory tissue is composed of the secretory units, called alveoli, responsible for the milk synthesis, and the connective tissue provides structural support for alveoli and contains blood and lymph vessels, and nerves.

In general, in locations where the animals have high production, milking is normally done twice daily. Several studies have corroborated an increase in yield with twice-daily milking when compared with once-daily milking (Papachristoforou et al. 1982; Capote et al. 1999; Boutinaud et al. 2003; Salama et al. 2003; Komara et al. 2009). Furthermore, the effects of the milking frequency on udder morphology (López et al. 1999), milk yield and quality (Capote et al. 1999), and milk partitioning and flow rate (Caja et al. 1999) were observed. However, to our knowledge, a detailed study that relates milking frequency with the tissue components in the caprine mammary gland has not been conducted.

In dairy goats, after colostrogenesis, the increase of prolactin (Castro et al. 2011) produces mammary growth and differentiation during early lactation, accounting for increasing milk yield during the ascending portion of the lactation curve, whereas after peak lactation, loss of mammary cells largely accounts for a declining milk yield (Capuco et al. 2001). So the dairy goat lactation is represented by an exponential secretory tissue development during the early lactation, produced by a combination of cellular hyperplasia and hypertrophy. In the declining lactation, a gradual involution due to an entirely reduced cell number is observed, and this occurs more rapidly after milking ceases (Knight and Wilde 1993). ELsayed et al. (2009) observed, in sections of goats’ mammary gland tissues, that the histological structure showed clear differences between lactation stages; being more developed at early and mid-stages, compared to late-stage of lactation.

The secretory rate is increased when milk is removed at a major daily milking session (Blatchford and Peaker 1982) due to milk being removed from the gland. In the same sense, Wilde et al. (1987) observed, after 37 weeks, greater amounts of RNA, DNA and several key enzymes in thrice-daily milking glands compared with twice-daily milking glands. In contrast, Stelwagen et al. (1994) described that after 36 h of milk accumulation, mammary tight junctions (TJ) had become disrupted, reducing synthetic activity and inducing apoptosis (Chedly et al. 2010; Argüello 2011). In dairy cows, several studies support the concept that serotonin (5-HT) is a feedback inhibitor of lactation (Hernandez et al. 2008). The mammary gland expresses multiple functional isoforms of serotonin receptors, and appears to be directly involved in milk protein gene expression (Hernandez et al. 2009). Finally, milk stasis in the bovine mammary gland induces 5-HT synthesis in the epithelium, where it affects TJ opening downstream of the 5-HT receptors (Marshall et al. 2010).

The cisternal udder compartment is very important in the milkability. The caprine cistern is able to accommodate a high level of milk production (Salama et al. 2003). Capote et al. (1999) indicated that the Majorera dairy goats have large cisterns, and this fact favours the lower milk losses (6%) if the goats were milked once-daily. Peaker and Blatchford (1988) reported several elevated cisternal milk percentages, ranging according to time elapsed after milking, from 66% (1 h) to 88% (16 h). Salama et al. (2004) described that the cisternal size at 24 h after milking (27.3±1.4 cm²) increased to 264% from 8 h after milking (11.1±1.3 cm²), and indicated that the caprine udder is more efficient at accommodating milk accumulation.

The objectives of the study reported were: (1) to elucidate the influence of the breed and milking frequency on the proportion of the different tissue components (secretory, ductal, connective and vascular) in the mammary gland in dairy goats and (2) to correlate the productive parameters (milk yield, milk composition, milk fractions and udder morphology) with the histological components.

2. Materials and methods

2.1. Animals and management

Experimental animal procedures were approved by the Ethical Committee of Las Palmas de Gran Canaria University. Thirty healthy goats, aged 3–4 years, on their third lactation were allotted in three groups, one for each breed (Majorera, Tinerfeña and Palmera). The herd was kept at the experimental farm of the ICIA (Instituto Canario de Investigaciones Agrarias, Tenerife, Spain) for six weeks during mid-lactation. The glands of the udder were milked in different frequencies from the tenth week of lactation to six weeks after. The management was as follows: the left gland was milked once daily (×1) at 0700 hours, and the right gland was milked twice daily (×2) at 0700 and 1700 hours. Goats were milked in a double 12-stall parallel milking parlour (Alfa-Laval, Madrid, Spain) equipped with recording jars (4 L) and a low-line milk pipeline. Milking was performed at a vacuum pressure of 42 kPa, a pulsation rate of 90 pulses/min and a pulsation ratio of 60/40 according to the milking parameters used in the breed (Capote et al. 2000). The milking routine included stripping the foremilk, machine milking, machine stripping before cluster removal and teat dipping in iodine solution (P3-cide plus; Henkel Hygiene, Barcelona, Spain). The animals were fed according to the Institute National de Recherche Agronomique (Paris, France) recommendations (Jarrige 1990).

2.2. Productive parameters testing

2.2.1. Udder morphology measures

The udder morphology measures were taken before the experimental period. Distance between teats (DT) was measured as the distance between the sphincters of the teats; cistern–floor distance (CF) and teat–floor distance (TF); and udder depth (UD) as the difference in distance between the udder insertion floor and the cistern floor.

2.2.2. Milk yield and milk-partitioning measures

During the 6 weeks of experimentation, once a week, to evaluate milk partitioning in the udder, according to Castillo et al. (2008), the goats were injected (i.v.) with 0.8 mg of OT receptor blocking agent (Atosiban, Ferring Lab., Mallmö, Sweden). The cisternal milk yield (CMY) for each udder half was recorded using the recording jars in the milking parlour. Afterwards, 2 IU of OT was injected (i.v.) to induce milk letdown. After 1 minute the alveolar milk yield (AMY) was recorded using the jars in the milking parlour. The total milk (TM) for each milking was calculated as the sum of the cisternal and alveolar milk. Using those data, the cisternal milk percentage (CMP) and the alveolar milk percentage (AMP) were estimated as the relation between cisternal and alveolar milk with the TM, respectively. To calculate the milk production per hour (HP), the TM in the a.m. milking was collected for each udder half and was divided by 24 and 14 h, for udder half milked ×1 and ×2, respectively.

2.2.3. Compositional milk analysis

The milk quality parameters measured were protein, fat and lactose percentages. Those parameters were analysed to the TM once a week, in the a.m. milking, using MilkoScan 133 (Foss Electric, Hillerod, Denmark).

2.3. Mammary tissue collection and staining procedures

Before sacrifice, each goat was weighed. The udder of each goat was removed and both glands were dissected from the teat to the dorsal parenchyma. Two samples from dorsal and medium parenchyma were randomly taken for each gland and were fixed in 10% neutral-buffered formalin, embedded in paraffin wax and sectioned at 4 µm. For the histological procedures, paraffin-embedded tissues were stained with haematoxylin and eosin to be examined by light microscopy.

2.4. Digital image acquisition and area measurements

Haematoxylin- and eosin-stained images of mammary tissue were captured with a top-mounted digital camera (Nikon D5000, Nikon Europe B.V., Spain) jointed to a microscope (Axiostar, Carl Zeiss Meditec Iberia S.A.U., Madrid, Spain). Nine pictures were consecutively taken at random locations in three zones within each tissue section, according to Castro-Alonso et al. (2010). These pictures were combined using Photoshop software (Adobe Photoshop CS4, Adobe Systems Incorporated, San José, CA, USA), which formed a huge picture where the different tissue components were presented. Digital images were transferred to a computer and stored for subsequent analyses.

All measurements were made by a digital image analysis program (Image-Pro Plus Version 4.5 for Windows; Media Cybernetics, Inc., Silver Spring, MD, USA). Areas occupied by the secretory tissue (alveoli), vascular tissue, ductal tissue and total tissue surface were determined for each image. This was achieved by using the computer mouse to outline desired structural features. The computer program recorded the area (µm²) for each structure that was outlined; these measurements were then exported to a Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) spreadsheet, where the total areas for each parenchyma component were calculated via summation. The area occupied by connective tissue was calculated by subtracting the total areas occupied by the secretory, vascular and excretory tissues from the total tissue surface. The alveoli area for each sample was estimated as the mean of all alveoli areas in each image. The number of alveoli in the sample was counted.

2.5. Statistical analyses

The results were expressed as percentages of tissue surface of secretory, ductal, connective and vascular components in each sample. A comparative analysis of the percentages of the different tissue surfaces in the three breeds was made using the analysis of variance (ANOVA), and the comparison of the percentages in the glands milked once daily or twice daily was made by Student's paired t-test. Differences between breeds and milking frequencies were considered to be significant at P < 0.05. To correlate the productive parameters with the histological components, Pearson's correlation coefficients were calculated. Analyses were performed using SPSS® 17.0 computer program (SPSS Inc. Headquarters, S. Wacker Drive, Chicago, IL, USA).

3. Results and discussion

3.1. Histological study

Macroscopic and microscopic observations of mammary gland showed a healthy mammary parenchyma without tissue damage produced by the different milking frequencies used in the study. In that sense, Salama et al. (2003) and Komara et al. (2009) did not observe changes in somatic cell counts due to once- or twice-daily milking. The microscopic study revealed the same histological structures as those observed by González-Romano et al. (2000), in which it was shown that in the same sample it could observe alveoli in different stages of lactation. So same alveoli presented abundant secretion in the alveolar lumen, and other presented a large lumen and depressed secretory cells, indicating the end of their activity.

Table 1 shows a comparative analysis of the means of the tissue parameters related to the breed and milking frequency. There were no interactions between breed and milking frequency. No effect of milking frequency on the parenchyma composition was observed. Based on the results obtained by Wilde et al. (1987) which needed an experimental period of 37 weeks to obtain greater concentrations of DNA, RNA and key enzymes in thrice-daily milking goats, we deduced that the experimental period (6 weeks) used in our study was not enough to produce changes in the tissue conformation.

Table 1. Effect of breed and milking frequency on tissue parameters and milk volume in udders of dairy goats.¹

				Milking frequency^2
	Majorera	Tinerfeña	Palmera	×1	×2
Tissue parameters	(n=34)	(n=35)	(n=35)	(n=53)	(n=54)
Secretory tissue (%)	47.63^a±5.86	43.94^b±6.69	45.40^ab±6.15	46.56±7.15	45.15±5.49
Connective tissue (%)	49.95^a±5.80	54.23^b±6.02	51.48^ab±5.82	51.33±6.58	52.31±5.59
Excretory tissue (%)	1.75±2.84	1.21±1.54	1.89±2.06	1.56±2.60	1.68±1.79
Vascular tissue (%)	0.67±0.56	0.62±0.53	1.23±1.65	0.87±1.41	0.82±0.57
Alveoli number	264.50^a±55	211.70^b±39.36	278.13^a±55.16	246.57±58.08	258.35±53.36
Alveoli area (µm^2)	17.13^ab±4.73	19.81^a±6.39	15.11^b±3.58	18.21±5.91	16.62±4.28
Note: Means with different superscript letters are statistically different (P < 0.05).
^1Data are the means±SD of all the samples for each breed and milking frequency.
^2Once (×1) or twice (×2) daily milking.

In contrast, the means of tissue parameters for Majorera, Tinerfeña and Palmera goats were significantly (P < 0.05) different. Majorera goat is the dairy goat with the highest percentage of secretory tissue (47.63%). With regard to the connective tissue, Tinerfeña goat has the highest means. The Palmera goat was situated in an intermediate position. These results could be explained by the fact that Tinerfeña and Palmera goats are adapted to mountainous and craggy zones, and that their udders have more connective tissue to support and protect the udder. The adaptation of the tissue components to a high productivity is very important in Majorera goats due to the arid environment in which this breed is raised. Therefore, the udder of this breed does not need a high percentage of connective tissue to avoid udder damage. The three breeds were affected by the dispersive effect of the genetic drift (island effect) of five centuries, producing different type of goats, all of them specialised as dairy animals (Martínez et al. 2006), living in a subtropical zone (Canary Islands) which have a high meteorological variation between islands.

3.2. Udder morphologic measures

Correlations between the udder morphological and histological measures are represented in Table 2. UD and distance between teats (DT) are related to the descent and the spherical nature of the udder, respectively. Combination of the both gives information about the globosity and length of the udder (the balance between the horizontal and vertical diameters). Capote et al. (2006) described the greater importance of the globosity of the udder than udder length measures with regard to milk yield and milking ability. In this sense, there was a positive correlation between UD and DT (r=0.46), which confirmed the excellent conformation of the udder in Canarian dairy goats. Table 2 shows UD and DT measures have positive and significant correlations with CMY, TM and HP). These results indicate that an increase in both parameters (UD and DT) produces bigger udders with better capacity to accommodate the milk production and cisternal compartments better for milk accumulation.

Table 2. Correlations between morphological and histological measures of the udder in dairy goats.

	DT	UD	CF	TF
UD	0.46
CF	0.13	−0.23
TF	0.31	−0.17	0.72**
CMY	0.53*	0.52*	−0.31	−0.26
CMP	−0.06	−0.32	−0.36	0.04
AMY	0.46	0.68**	−0.05	−0.23
AMP	0.06	0.32	0.36	−0.04
TM	0.53*	0.58*	−0.26	−0.26
HP	0.64**	0.68**	−0.15	0.16
Fat	0.13	−0.19	0.39	0.11
Protein	0.05	−0.15	0.35	0.18
Lactose	0.44	0.45	0.46	0.37
Weight	0.35	0.54*	−0.56*	−0.35
Secretory tissue	−0.18	0.41	0.22	−0.20
Connective tissue	0.17	−0.38	−0.32	0.15
Excretory tissue	0.11	−0.30	0.48*	0.30
Alveoli area	−0.33	−0.10	−0.50*	−0.62**
Alveoli number	0.35	0.32	0.67**	0.53*
DT, distance between teats; UD, udder depth; CF, cistern–floor distance; CMY, cisternal milk yield; CMP, cisternal milk percentage; AMY, alveolar milk yield; AMP, alveolar milk percentage; TM, total milk; HP, milk production per hour.
*P < 0.05; **P < 0.01.

Cistern–floor distance (CF) and teat–floor distance (TF) provide information about the descent of the udder. In addition, the difference between distances affect in a high yield of machine stripping milk, the portion of the cisternal milk which remains under the teat (López et al. 1999). No significant correlation with UD was detected. Capote et al.'s (2006) remark on this may indicate a greater importance of the globosity of the udder compared with length parameters. There is also a positive correlation between CF and TF. The weight of the goat and the CF are correlated (r= − 0.56), indicating that the largest goats have bigger and more descended udders. Alveoli area and alveoli number are negatively and positively correlated, respectively, with the length parameters (CF and TF). Explained by the fact that less descended udders have smaller alveoli, and a greater number of alveoli in the same area.

3.3. Milk yielding and milk partitioning measures

Table 3 shows the correlations between milk yield and fractioning and histological measures. CMY and AMY are positively correlated with the milk-yielding parameters (TM, r=0.99 and r=0.86; HP, r=0.53 and r=0.50, for cisternal and alveolar milk quantity, respectively). These results are in concordance with Castillo et al. (2008), who found these correlations in dairy ewes (TM at 16 h, r=0.89 and r=0.70; TM at 24 h, r=0.93 and r=0.68, for cisternal and alveolar milk, respectively).

Table 3. Correlations between milk yielding and fractioning and histological measures in dairy goats.

	CMY	CMP	AMY	AMP	TM	HP
CMP	0.05
AMY	0.77**	−0.57*
AMP	−0.05	−1.00**	0.57*
TM	0.99**	−0.10	0.86**	0.10
HP	0.53*	−0.09	0.50*	0.09	0.54*
Fat	−0.04	−0.47*	0.22	0.47*	0.02	−0.16
Protein	−0.24	−0.28	−0.03	0.28	−0.20	−0.42
Lactose	0.25	−0.23	0.38	0.23	0.29	0.20
Weight	0.43	0.30	0.22	−0.30	0.40	0.46
Secretory tissue	−0.12	−0.48*	0.25	0.48*	−0.04	−0.15
Connective tissue	0.16	0.54*	−0.24	−0.54*	0.07	0.18
Excretory tissue	−0.15	−0.04	−0.18	0.04	−0.16	−0.14
Alveoli area	−0.06	0.34	−0.25	−0.34	−0.11	−0.30
Alveoli number	0.09	−0.62**	0.45	0.62**	0.18	0.38
CMY, cisternal milk quantity; CMP, cisternal milk; AMY, alveolar milk quantity; AMP, alveolar milk; TM, total milk; HP, milk production per hour.
*P < 0.05 **P < 0.01.

The percentages of the milk partitioning (CMP and AMP) have no correlation with the milk-yielding parameters. The percentage of fat contained in the milk is positively correlated with AMP due to the alveolar milk being richer in fat (Capote et al. 1999; Ayadi et al. 2004; Castillo et al. 2008). These percentages present correlations with the main tissue components (secretory tissue, r=0.48, connective tissue, r= − 0.54, for alveolar milk percentage). This is due to the possibility of carrying more milk if the parenchyma has a high percentage of secretory tissue. This is also the case with the alveoli number; a higher number of alveoli make a greater accumulation of milk in the alveolar compartment possible.

3.4. Compositional milk analysis

The correlations between milk composition and histological measures are represented in Table 4. Lactose percentage only presents a significant correlation with the number of alveoli. This is due to the fact that the greater number of alveoli carries a greater number of epithelial cells, the only cells in the body with the capacity to synthesise lactose (Stelwagen et al. 1994). Fat percentage is positively correlated with the excretory tissue and alveoli number, due to alveoli and intralobular ducts (ductal tissue) containing alveolar milk, which is rich in fat.

Table 4. Correlations between milk quality and histological measures in dairy goats.

	Fat	Protein	Lactose
Protein	0.14
Lactose	0.18	0.35
Weight	−0.66**	−0.02	0.01
Secretory tissue	−0.20	0.26	0.28
Connective tissue	0.06	−0.28	−0.30
Excretory tissue	0.70**	0.04	0.13
Alveoli area	−0.55*	0.12	−0.32
Alveoli number	0.47*	−0.09	0.48*
*P < 0.05; **P < 0.01.

4. Conclusions

First of all, the results about the milking frequency influence on the tissue components were not different; probably the experimental period was short. The tissue composition in udders of the three dairy goat breeds varies due to the fact that each breed is adapted to a different environment and management, and centuries of isolation in the islands contributed to the genetic variance. The study of the correlation between the productive parameters and tissue components demonstrated that the morphological udder characteristics, in particular the globosity (represented by the combined effect of UD and DT), determined an udder with high yields. Otherwise, in accordance with the findings of other authors, the cisternal compartment in goats has a greater importance in the dairy capacity. Finally, the percentages of both secretory and connective tissues seem not to have an influence on the milk yield in high yielding dairy goats.

Acknowledgements

We are grateful to Patricia Barber (Department of Quantitative Methods in Economics and Management, Las Palmas de Gran Canaria University) for her assistance with the statistical analyses.