Levels of lactose from both infused and uninfused udder halves were normal ... Right udder halves of control animals (n = 2) were ..... a better understanding.
hr. J. Biochem. Vol. 16, No, 11, pp. 113s1141. 1984 Printed in Great Britain. Al1rights reserved
0020-71 IX.‘84 ~3.~~+0.~ Pergamon Press Ltd
Copyright :c: 1984
EFFECTS OF COLCHICINE ON MILK YIELD, COMPOSITION, AND CELLULAR DIFFERENTIATION DURING CAPRINE LACT~GEN~SIS* LORRAINE M. SORDILLO~,STEPHEN P. OLIVER& ROBERTT. DUBY and Rosy~ RUFNER Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, U.S.A. (Receioed
24 January
1984)
Abstract-1, ~nt~drn~m~ry colchicine infusion into goats at parturition reduced milk yield by 20:; during the 30 day experimental period. 2. During the first week of lactation, milk composition from colchicine-treated udder halves had elevated somatic cell numbers. serum albumin concentration and pW, while citrate concentration was lower in comparison to uninfused glands. 3. Levels of lactose from both infused and uninfused udder halves were normal during the first week of lactation. 4. No differences were observed in degree of alveolar development in tissue samples collected prior to treatment. 5. Light and electron microscopy suggested that colchicine-treated udder halves consisted predominantly of undi~erentiated mammary secretory cells, while uninfused udder haivcs anoeared more 1I cytologically differentiated. 6. Results demonstrated that intramammary cofchicine infusion at parturition temporarily altered milk composition and inhibited ~rnrna~ cellular differentiation.
INTRODUCTION The plant alkaloid, colchicine. interferes with release of secretory products in a variety of tissues including insulin by the pancreas (Lacy and Malaisse, 1973). plasma proteins from the liver (Redman ez ai., 1975), catecho~~mines from the adrenal medulla (Poisner and Bernstein, 1971) and hormone secretion by the thyroid gland (Wolff and Williams, 1973). Data suggest that microtubules are involved in these secretory mechanisms and that coichicine d&aggregates the cytoskeletal network into smaller tubulin subunits (Mareel et ul., 1980). Colchicine has been extensively used to study mechanisms of milk synthesis and secretion. Intramammary infusion of colchicine into lactating goats produced a reversible reduction in milk yield with concurrent inhibition of casein and fat globule secretion (Patton, 1976). Lactating mammary cells from rats and cows treated both in h-o and in ciao with colchicine exhibited reduced secretion of fat (Morales et al., 1982), lactose (Guerin and Loizzi, 1977, 1978), and protein (Smith et a/., 1982). Colchicine also inhibited milk synthesis and altered ionic composition by inhibiting mammary substrate uptake and blood flow in goats (Henderson and Peaker, 1980). Ultrastructural observations of lactating mam*Approved as Journal Article No. 2635, Massachusetts Agricultural Experiment Station, Universitv of Massachusetts, Amherst, U.S.A. tPresent address: Hill Farm Research Station. Louisiana Agricultural Experiment Station, LSU Agricultu~i Center. Rt 1, Box IO, Homer, LA 71040, U.S.A. $Author to whom reprint requests should be addressed.
mary cells suggested that colchicine interfered with secretory vesicle fusion with plasma membranes (Knudson et al., 1978). Colchicine may also disrupt intracellular microtubuiar integrity necessary for exocytotic mechanisms (Nickerson et al., 1980; Reaven and Reaven, 1977). In an attempt to acceferate involution, colchicine infused into bovine mammary glands in late lactation caused significant reductions in milk yield and altered secretion composition (Oliver and Smith, 1982a). Prepartum intramammary infusion of colchicine resulted in reduced rates of fatty acid synthesis, CO? production, and protein synthesis in tissue explants while also markedly reducing subsequent milk production in vim (Akers and Nickerson, 1982; Akers and Nickerson, 1983). Histological and cytological evidence support the contention that prepartum colchicine treatment irreversibly suppressed differentiation of mammary epithelia (Nickerson and Akers, 1983). Lactogenesis involves a cascade of events whereby mammary cells begin to synthesize and secrete copious quantities of milk. Understanding mechanisms which regulate cellular differentiation and the onset of lactation need to be further defined and may provide new approaches for increasing milk production. Studies relating mammary cell structure to biochemical events have provided valuable information regarding alveolar function during lactation. However, studies using these techniques during lactogenesis are minimal. This study reports effects of intrammammary colchicine infusion at parturition on milk yield, composition, and cehular differentiation in the caprine mammary gland.
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LORRAINE M. SORDILLO et al.
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MATERIALS AND METHODS Dairy goats of mixed breeding from the University of Massachusetts research herd were used in this experiment. A stock solution of colchicine (1 mg/ml) was prepared in physiological saline and sterilized through a 0.45 pm Millipore filter. Right udder halves of 5 goats were intramammarily infused immediately after parturition with 5 ml colchicine stock solution emulsified with 5ml Freund’s incomplete adjuvant (Difco Laboratories, Detroit, Michigan). Right udder halves of control animals (n = 2) were infused with an emulsion containing 5 ml Freund’s incomplete adjuvant and Sml sterile physiological saline. Left udder halves of all animals were not infused and served as within animal controls. Goats were milked twice daily at 12 hr intervals and yields were recorded throughout the experimental period. Milk samples for compositional analysis were collected from all udder halves immediately prior to infusion and 2, 4, 7, 14 and 30 days postparturition. Skim and whey fractions were prepared for compositional analysis as outlined by Oliver and Smith (198213). The direct microscopic cell count method (Schultze et al., 1968) was used to determine number of somatic cells in milk samples. Lactose was determined calorimetrically as described by Teles et al. (1978). Citrate was quantified by a modification of the White and Davies (1963) method. Skim milk (0.15 ml) was added to disposable test tubes containing 1.35 ml deionized distilled water and 1.5ml 24% trichloroacetic acid. After 30 min incubation at room temperature, tubes were centrifuged at 124Og for 30min. The supernatant (0.5 ml) was added to clean test tubes followed by the addition of 0.65 ml pyridine and 2.85 ml acetic anhydride. After thorough mixing between addition of reagents, each tube was stoppered and incubated in a 32°C water bath for 30min. Color development was measured at 428 nm. Serum albumin was determined by electroimmunodiffusion as described by Schanbacher and Smith (1974). Rabbit anti-goat serum albumin and purified caprine serum albumin were purchased from Sigma Chemical Co., St Louis, Missouri. Tissue samples for histological and cytological examination were obtained by mammary biopsy. One goat was biopsied from both colchicine treated and control udder halves prior to infusion and again 7 days postpartum. The
:
2
.m x.
Fig.
_--
.__-_-.---
/
1. Changes in udder half milk yield following mammary infusion of colchicine at parturition.
Intrarnammary infusion of colchicine at parturition reduced milk production by 20% in treated udder halves compared to uninfused controls (Fig. 1). The greatest disparity in milk yields occurred in the first 4 days of lactation. Although the degree of inhibition decreased as lactation progressed, colchicine-infused udder halves never reached the production level attained by uninfused halves.
in caprine mammary secretion composition between colchicine-infused udder halves throughout the 30 day period following treatment
and uninfused
Days after treatment*
Udder half
0
2
4
I
14
30
Treated Control
6.61 6.43”
6.93” 6.61”
6.90” 6.63”
6.56” 6.42”
6.30” 6.18”
6.33” 6.26”
Treated Control
I .46h
5.82
5.17’
0.81”
0.40”
0.29”
1.12”
0.98’”
0.47”‘r
0.64”
0.40“
0.23”
Citrate (mgiml)
Treated Control Treated Control
6.41” 6.44 1.16’ 1.13”
6.55 6.57” l.llh 1.12”
6.70” 6.54”t 0.59” 1.03at
6.66 6.56” 0.76y” 0.96”
6.64” 6.62” l.16h 1.16”
6.67” 6.60” l.lgh I .28
Lactose (mg/mU
Treated Control
45.5” 55.1”
59.2nh 62.90h
68.9h 68.8Yh’
58.1Qh 58.9””
60.3”h 84.7
74.1” 76.4
Parameter Somatic
cells
(lo&o) Serum albumin (mgiml) PH
intra-
RESULTS
deep incision was made through the skin above the gland cistern and approximately 1 cm’ of tissue was removed. The incision was sutured with cat gut and antibiotics applied topically. Excised tissue samples were immediately placed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.0 at 37°C) and diced into l-2 mm3 portions. Tissue I. Changes
loo-
was then fixed and dehydrated (Warchol et al., 1974), and embedded in BEEM capsules with Epon 812 (Luft, 1961) or Spurr’s medium (Spurr, 1969). Ultrathin sections approximately 60 nm thick were obtained on a LKB Ultratome III ultramicrotome, stained with 5.0% uranyl acetate in 509,, methanol for 20min followed by 0.494 lead citrate for 20min, and examined with a Zeiss EM 9S-2 transmission electron microscope at 60 kV. Thick sections (0.5-I pm) were also obtained and stained with Toluidine Blue for light microscopy. Quantitative morphological analysis determined percentage mammary tissue area composed of interalveolar connective tissue, epithelia, and alveolar lumen. For each of the 4 biopsy samples, 5 slides containing thick sections were randomly selected for classification. Six replications of 35 contact points were counted per slide at a magnification of 400x (Nickerson and Akers, 1983). A split-block experimental design was used to compare differences in secretion composition between colchicineinfused vs uninfused udder halves for all sampling periods. Data were statistically analyzed by a general mixed model analysis of variance with equal cell sizes.
goat was anesthetized prior to surgery with sodium pentabarbital (50 mg/ml) via an indwelling catheter. A 2.5 cm
Table
:$=-5 _--
*Treated halves were infused with 5 mg colchicine at parturition; control halves were not infused. Mammary secretion obtained prior to experimental manipulation is represented by day 0. “~*~‘Means within treatment halves lacking identical superscript differ (P i 0.05). tMeans between halves within sample period differ (P < 0.05).
Fig. 2. (a) Mammary tissue obtained at parturition prior to experimental manipulation. x 800. (b) Mammary cells at parturition illustrating a large cytoplasmic to nuclear ratio, numerous supranuclear vacuoles, and basally located nuclei. x 2000. (c) Mammary tissue from uninfused-udder halves obtained 7 days postpartum demonstrating decreased interalveolar stromal area and increased alveolar luminal area in comparison to Fig. 2a. x 800. (d) Mammary epithelium from uninfused-udder halves at 7 days postpartum exhibiting polarized cells with abundant cytoplasmic vacuoles, apically located fat droplets, and basally located spherical nuclei. x 2000. (e) Mammary tissue from colchicine-treated halves at 7 days postpartum demonstrating reduced luminal area and increased interalveolar stroma. x 800. (f) Mammary epithehum from colchicine-treated udder halves at 7 days postpartum exhibiting undifferentiated cell characterized by the presence of large fat droplets scattered about the cytoplasm, absence of secretory vacuoles, and medially located irregularly shaped nuclei. x 2000. Symbols used: F-fat droplet; L--lumen; N-nucleus; SV-secretory vesicles; S-stroma.
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Fig. 3. (a) Representative mammary epithelium from control tissue obtained 7 days postpartum. Fully differentiated secretory cell characterized by hypertrophic Golgi (G), extensive RER network (R), and casein containing secretory vesicles (SV) in the supranuclear region. x 5700. (b) Representative cell type observed in colchicine-treated tissue obtained 7 days postpartum. Undifferentiated mammary epithelial cell characterized by irregularly shaped and medially located nuclei (N), with accumulation of large fat droplets throughout the cytoplasm. x 5,700. Symbols used: F-fat droplet; L-lumen; S-stroma.
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Caprine lactogenesis Table 2. Morphometric analysis of mammary tissue from colchicine-infused and uninfused udder halves at parturition and 7 days postpartum Biopsy period Characteristic”
Udder half
Parturition”
Day 3
Epithelium
Treated Comrol
35.0 i 2.85 35.0 * 2.82
40.6 + 1.92 32.9 f 2.41
Stroma
Treated Control
21.0 k 2.23 20.0 i 2.16
44.0 k 3.29 15.7 + 2.68
Lumen
Treated Control
44.0 k 3.29 44.9 & 3.65
15.4 i 2.28 51.4i4.65
“Data expressed as mean percent k SD of total tissue area.
‘Tissue obtained prior
to experimental
Changes in milk composition in colchicine-infused and control haives are found in Table 1. Increased somatic cell numbers, serum albumin, and pH were observed in milk from colchicine-infused halves during the first week of lactation. The greatest increases were detected by 4 days of lactation when milk from infused glands had an average of 6.90 log,,/ml SOmatic cells, 5.77 mg/ml serum albumin, and a pH of 6.70. By day 30 postpartum, milk composition was similar in both infused and control udder halves. Citrate concentration followed similar trends. There were no significant differences in citrate between udder halves prior to treatment. However, by day 4 postinfusion, colchicine-infused udder halves had lower levels of citrate whereas in control halves, citrate concentration remained relatively constant. Changes in citrate concentrations were transient since levels returned to normal by 7-14 days postpartum. In contrast to other parameters measured, no differences in milk lactose concentration were observed between udder halves during the first week of lactation. No differences in milk yield or composition were observed between udder halves in the 2 control animals infused with saline and adjuvant only. Morphological analysis of both colchicine-infused and control udder halves (Table 2) was consistent with milk composition data (Table 1). No differences in degree of secretory cell development were observed in tissue samples collected at parturition, prior to infusion (Figs 2a and b). Total epithelial area in both udder halves increased by day 7 postpartum (Figs 2c-f). However, colchicine-treated mammary tissues exhibited more interalveolar stroma (+ 28.3%) and less luminal area (-36.0%) compared to control tissue (Table 2). Although percent total alveolar epithelium between udder halves was not different, cytological observation indicated considerable variation in development. Mammary cells from uninfused udder halves were polarized with basally located nuclei, apically located fat droplets and abundant cytoplasmic vacuoles (Fig. 3a). Cells from colchicine-treated glands exhibited minimal cytoplasmic area, lack of cellular polarity, irregularly shaped and medially located nuclei, and randomly located fat droplets (Fig. 3b). DISCUSSION
Intramammary infusion of colchicine at parturition suppressed subsequent milk production by 20%. Milk yields in colchicine-treated halves gradually increased over the 30 day experimental period
manipulation.
but did not approach control levels (Fig. I). This is in contrast with previous studies in which intramammary infusion of colchicine into lactating goats resulted in a significant but reversible depression in milk yield (Patton, 1976). Although the majority of lobulo-alveolar growth in goats occurs in the last trimester of pregnancy (Knight and Peaker, 1982), studies suggest that development continues until approximately one week postparturition (Anderson et al., 1981). Since colchicine is a potent mitotic inhibitor (Margolis and Wilson, 1978), it may be inferl;ed that mammary secretory cells undergo postparturient mitosis critical to lactation. Alternatively, it has been suggested that prepartum intramammary infusion of colchicine disrupts microtubules necessary for prolactin-induced cellular differentiation of mammary secretory cells (Akers and Nickerson, 1983). Prior to treatment, tissue was morphologically equal in proportions of alveolar epithelia, alveolar lumens, and interalveolar connective tissue (Table 2). However, mammary tissue obtained 7 days postpartum from colchicine-treated halves exhibited a reduction in secretory activity displaying less luminal area and more connective tissue stroma. In addition, epithelial cells from treated halves expanded at the expense of alveolar lumina due to the accumulation of large fat droplets in the cytoplasm (Fig. 3b). Although the percent total alveolar area for both biopsies was approximately equivalent between udder halves, control tissue appeared more differentiated, exhibiting extensive organelle development. Furthermore, the capacity of control tissue to actively secrete milk was more pronounced, e.g. alveolar lumens were distended and stromal tissue was displaced. These findings are consistent with ultrastructural observations from prepartum colchicine-treated tissue in the bovine mammary gland (Nickerson and Akers, The interruption of mammary 1983). cell differentiation in treated tissue at parturition parallels observed reductions in milk yield (Fig. 1). Together, these data support the possibility of considerable postparturient secretory cell division (Franke and Keenan, 1979) and growth (Anderson et al., 1981) in the mammary gland. Intramammary infusion of colchicine resulted in an inflammation that was detectable by udder palpation. Mammary secretion composition also indicated an inflammatory response with elevated somatic cell numbers, serum albumin, and pH by 24 hr posttreatment. Levels of somatic cells in treated glands gradually increased to an average of 6.90 log,,, cells
LORRAINE M. SORDILLO et al
1140
per ml by day 4 of lactation. This well exceeds levels found in normal goat milk during early lactation (Pettersen, 1981). Previous work suggested that elevated levels of somatic cells coincided closely with breakdown of mammary tissue and increased permeability of tight junctions between epithelial cells (Kitchen, 1981). In support of this hypothesis, levels of serum albumin in treated udder halves rose in conjunction with somatic cells (Table 1). The majority of serum albumin in goat mammary secretion is thought to be derived passively from blood with approximately 10% acquired by de novo synthesis (Phillippy and McCarthy, 1979). Therefore, increased levels of serum albpmin suggest that colchicine infusion resulted in some form of tissue trauma allowing serum albumin secretion into milk via a paracellular route. Levels of citrate in colchicine-treated udder halves were significantly lower than controls throughout the first week of lactation. Citrate in milk is believed to be formed within mitochondria and secreted by secretory vesicles derived from the Golgi (Faulkner and Peaker, 1982; Zulak and Keenan, 1983). Microtubules associated with the Golgi are involved in transporting vesicles to the plasma membrane (Nickerson et al., 1980; Falconer, 1980). The temporary reduction in citrate may be explained by colchicineinduced disruption of microtubules necessary for the exocytotic process. Lactose is formed in the Golgi and packaged in secretory vesicles as well. Unlike citrate, however, we observed no differences between udder halves in lactose concentration. Patton (1976) also found levels of lactose unaffected by intramammary colchicine infusion in lactating goats. Conversely, in vitro data revealed that colchicine inhibited synthesis and secretion of lactose in guinea pig mammary slices (Guerin and Loizzi, 1977, 1978). Colchicine infusion during the onset of lactation may provide valuable insight regarding lactogenic responses of the mammary gland. A successful lactation not only requires proliferation of alveolar cells during pregnancy but also adequate cellular differentiation to acquire synthetic and secretory capabilities. Previous studies utilizing intramammary colchicine infusion emphasized the importance of microtubules for mammary cell differentiation. However, results of the present study suggest that the inflammatory properties of colchicine are partially responsible for the actions on cellular processes. Observed decreases in milk yield and citrate in conjunction with increased levels of serum albumin, somatic cells, and elevated pH are indicative of an inflammatory response. Similarly, the histological alveolar imply interrupted observed changes differentiation and possible disruption of mammary cell integrity. Further studies on the actions of colchicine during lactogenesis could aid in establishing the relationship between form and function leading to a better understanding of physiological events crucial to the subsequent lactation. REFERENCES Akers R. M. and Nickerson S. C. (1982) Effect of periparturient disruption of mammary cell microtubules on initiation of milk secretion in heifers. J. Dairy Sci. 65, 84.
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Metabolic energy and cytoskeletal requirements for synthesis and secretion by acini from rat mammary gland--l. Ultrastructural and biochemical aspects of synthesis and release of milk proteins. I~I. J. Biochem. 14, 87-98. Spurr A. R. (1969) A low viscosity epoxy embedding . medium for electron microscopy. J. (/l/r& R>s. 26, 3 1-43. Teles F.. Feitosa F.. Young C. K. and Stall J. W. (1978) A method for the rapid determination of lactose. J. Dairy Sci. 61, 506-508. Warchol J. B., Herbert D. C. and Rennels E. G. (1974) An improved fixation procedure for microtubules and microfilaments in cells of the anterior pituitary. Am. .I. Anat. 141, 427432. White J. C. D. and Davies D. T. (1963) The determination of citric acid in milk and milk sera. J. Dairy Res. 30, 171-189. Wolff J. and Williams J. A. (I 973) The role of microtubules and microfilaments in thyroid secretion. Rec. Propr. Horm. Res 29, 229-285. Zulak I. M. and Keenan T. W. (1983) Citrate accumulation by a Golgi apparatus rich fraction from lactating bovine mammary gland. Int. J. Biochem. 15, 747-750.
