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Visceral Tissue Growth and Proliferation During the Bovine Lactation Cycle^*,

¹ Growth Biology Laboratory and
² Gene Evaluation and Mapping Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705-2350

Corresponding author: Ransom L. Baldwin, VI; e-mail: rbaldwin{at}anri.barc.usda.gov.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Twenty one multiparous, nonpregnant, lactating dairy cows were used to assess the impact of stage of lactation on visceral tissue mass and small intestinal cell proliferation. Cows were slaughtered at each of 4 stages of lactation: 14, 90, 120, and 240 d of lactation. With stage of lactation, DMI increased through d 90 and thereafter remained similar through d 240 (quadratic). Carcass weight and empty body weight (EBW) declined with stage of lactation through d 120 and increased thereafter (quadratic). As a percentage of EBW, rumen, small intestine, and liver weights increased with increasing stage of lactation (quadratic), increasing from 14 to 120 d and declining through 240 d. Stage of lactation did not have a measurable affect on reticulum, omasum, abomasum, or large intestine weights as a percentage of EBW. Visceral adipose mass as a percentage of EBW declined with stage of lactation to a minimum at 120 d and increased by 240 d (quadratic). Concentrations of RNA and DNA of digestive tract organs were largely unaffected by stage of lactation with the exception of the liver DNA concentration through d 120 (quadratic). The proliferative growth fraction (Ki67) was unaffected by stage of lactation. However, bromo-deoxyuridine labeling of jejunal crypts exhibited a cubic response with stage of lactation and tritiated thymidine incorporation by duodenal epithelium increased with stage of lactation through d 120, declining thereafter (quadratic). Mass of visceral tissues increase to meet the energetic demands of lactation and that increased absorption capacity of the intestines is achieved by hyperplastic growth of the intestinal epithelium.

Key Words: lactation • dairy • cow • visceral organs

Abbreviation key: BrdU = bromodeoxyuridine, EBW = empty body weight, ME = metabolizable energy, NGS = normal goat serum, TBS = Tris-buffered saline

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
In the ruminant, visceral tissues account for 30 to 50% of whole-body heat energy loss, and thus have a considerable impact on the partitioning of metabolizable energy (ME) between heat energy loss and net energy for lactation. Although fasting has been demonstrated to decrease mass specific metabolic activity (McBride and Milligan, 1985), the majority of oxidative energy use is related to maintenance of tissue integrity and mass (Gill et al., 1989; McBride and Kelly, 1990). Thus, specific nutrient metabolism (Baldwin and McLeod, 2000) and tissue O₂ consumption (McLeod and Baldwin, 2000) are largely unaffected by alterations in nutrient delivery in the ruminant. Moreover, the increase in nutrient metabolism by the intestinal tissues that is observed when the energy density of the diet is decreased is primarily attributable to increases in gastrointestinal epithelial cell mass (McLeod and Baldwin, 2000).

Undoubtedly, the most dramatic physiological increase in gut tissue metabolism and growth in the dairy cow occurs during early lactation as ME intake increases from slightly above maintenance to near 4 times maintenance. The onset of lactation results in a substantial decline in BW as fat is mobilized from adipose tissue, until the size and efficiency of the digestive tract of the cow can respond to the increased nutrient demands imposed by lactation (Mayer et al., 1986). Smith and Baldwin (1974) found increases (20 to 31%) in liver and gut tissue mass in lactating cows relative to nonlactating cows presumably due to both physiological and nutritional factors. Moreover, similar alterations in tissue mass are also seen in other service-oriented tissues (e.g., kidneys, liver, heart and lungs; Johnson et al., 1990) of ruminant animals and in the gastrointestinal tract of other species (Fell et al., 1963; Canas et al., 1982; Ferrell and Koong, 1986) in response to changes in physiological state and energy intake.

Change in tissue mass is the net result of rates of cellular proliferation, and cellular losses, as well as changes in cellular size. In lactating dairy cattle, the proliferative or hypertrophic response of the visceral tissues to the nutritional demands imposed by lactation are not well defined. This study was conducted to better define the growth response of the visceral organs through lactation.

	MATERIALS AND METHODS

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Experimental Protocol
The animal protocol for this research was approved by the Beltsville Agricultural Research Center Institutional Animal Care and Use Committee. Twenty multiparous, nonpregnant, clinically normal, lactating Holstein cows from the Beltsville Agricultural Research Center dairy herd were used. Cows were housed in tie stalls with 12 h of light/d and were milked twice daily at 0700 and 1800 h. Four to 6 cows were slaughtered at each of 4 stages of lactation: 14 DIM (4 cows), 90 DIM (5 cows), 120 DIM (6 cows), and 240 DIM (6 cows). Because cows were concurrently participating in a mammary gland physiology study (Capuco et al., 2001), all cows except the 14 DIM group were synchronized with 2 injections of prostaglandin F₂ (Lutalyse, The Upjohn Co., Kalamazoo, MI) 14 d apart. Cows were killed 14 d after the second injection. During the 24-h period prior to slaughter, each cow received four injections of bromodeoxyuridine (BrdU; Sigma Chemical Co., St. Louis, MO) via indwelling jugular catheter at 7.5-h increments, allowing for 22.5 h between the first and last injection. Cows were slaughtered 1 to 2 h following the final injection. Bromodeoxyuridine was administered at 2.25 mg/kg of BW per injection (Yanai et al., 1996) at a concentration of 20 mg/mL of 0.9% saline (pH 8.2).

Feeding and Feed Chemical Analysis
Cows were provided ad libitum access to fresh water and were fed a common silage-based TMR (Table 1) once daily. Diets were formulated to meet or exceed the requirements for RDP, RUP, NDF, ADF, Ca, and P for lactating cows (NRC, 1989). With the exception of the 14 DIM group, all cows were fed the common lactation ration for at least 21 d prior to slaughter. Following parturition, fresh cows were transitioned from a pre-fresh ration (50% dry cow diet with 50% lactation diet) onto the common experimental ration by 4 DIM. Total mixed rations were prepared each day, and daily samples of silage (orchardgrass, alfalfa, and corn silages) and grain mix were collected, and DM content (100°C) was determined. Average DM was calculated weekly and used to adjust diets and the amount of feed offered the following week. Feed offered and orts were collected and recorded daily using the Calan Gate and Data Ranger system (Calan Data Ranger; American Calan Inc., Northwood, NH). Samples (500 g) of the TMR and orts were collected daily, composited weekly, dried to a constant weight (55°C) for determination of DMI. Composite TMR, and orts were dried (55°C), and ground using a 1-mm screen (Centrifugal Grinder model ZM1, Brinkmann Instruments Co., Westbury, NY) prior to analysis for fiber and ash content (AOAC, 1975). Wet composite samples of TMR and orts were analyzed for N and C content (LECO C/N 2000; model 601-900-000; LECO Corporation, St. Joseph, MI), and gross energy content by combustion in an adiabatic bomb calorimeter (model 1241; Parr Instrument Co., Moline, IL) with the aid of a polyethylene primer bag of known weight and energy content. Fiber analysis was conducted using the detergent analysis system (Goering and Van Soest, 1970) adapted for the ANKOM²⁰⁰ fiber unit (ANKOM Technology Corporation, Fairport, NY). The NDF procedure was modified by the addition of heat-stable amylase (Sigma Chemical Co.) to all samples during analysis.

Small and large intestines were separated from the mesentery, rinsed extensively with 4 to 6 L of saline (0.9% NaCl; 37°C), and weighed. Lengths were determined by looping the intestine across a stationary board, fitted with plastic pegs at 1-m increments, without tension to minimize stretching. One-meter intestinal sections from the duodenum (1 m distal to the pyloric sphincter), jejunum (midpoint of the small intestine), and ileum (1 m anterior to the ileocecal junction) were weighed and used for further subsampling. Two sections (10 cm) were taken from each of the 1-m intestinal sections, weighed, and processed for total tissue and epithelial composition analysis. Epithelial tissue was harvested by splitting one of the 2 10-cm sections along its longitudinal axis and scraping with a glass slide. Immediately after weighing, rumen epithelium and small intestine, both total and epithelial, were partitioned for RNA and DNA (0.3 to 0.5 g frozen in 2 mL of ice cold extraction buffer, 10 mM Tris with 5 mM EDTA, pH 8.0, with dry ice), DM (100°C for 48 h), and N (carbon/nitrogen analyzer, LECO). For immunohistochemical analyses, representative samples were placed in 10% neutral buffered formalin (Sigma HT50-1-128). All visceral organs and tissues were weighed, processed, and (or) frozen within 45 min of exsanguination.

DNA and RNA Analysis
Frozen samples were diluted further to 1:14 sample-to-buffer ratio in ice cold extraction buffer and immediately homogenized (model PT10/35, Brinkmann Instrument) on ice by applying 3 1-min bursts over 5 min. A 1.0-mL aliquot of each tissue homogenate was placed into a 1.5-mL polypropylene microcentrifuge tube and frozen (–70°C) for DNA analysis using the Hoechst 33258 dye binding procedure of Labarca and Paigen (1980) modified for a 96-well microtiter plate assay using a Fluorometer (FL600, Bio-Tek Instruments, Inc., Highland Park, Winooski, VT). A second 1.0-mL aliquot was placed into a 2.0-mL microcentrifuge tube and assayed for RNA using the method described by Schmidt and Thannhauser (1945).

[³H-]Thymidine Incorporation Assay
Duodenal epithelium (approx. 100 mg wet wt) scraped from a 10-cm segment cut from 2 m distal to the pyloric sphincter, was incubated in triplicate flasks containing Medium 199 (3 mL) with 1 µCi of ³H-thymidine/mL. Incubations were conducted for 2 h at 37°C in a shaking water bath, under an atmosphere of 5% CO₂ and 95% O₂. After incubation, tissue was rinsed in 0.9% saline and frozen (–20°C). Frozen samples were subsequently homogenized in saline and precipitated with TCA, and ³H-thymidine incorporation was determined by liquid scintillation counting as previously described (Capuco and Akers, 1990).

Immunohistochemistry Procedures
Tissue pieces, fixed in 10% neutral buffered formalin for 24 h post-slaughter, were transferred to 70% ethanol, stored, and sent to American Histolabs, Inc. (Gaithersburg, MD) for paraffin embedding and sectioning. Fixed tissue sections were stained using the microwave antigen retrieval procedures of Garrett and Guthrie (1998). Briefly, sections were placed in xylene to remove the paraffin, rehydrated through a descending alcohol series, and then rinsed in deionized water (3 x 2 min). Sections were placed into 10 mM citrate buffer (pH 6) for microwave antigen retrieval via microwave oven (General Electric; 800 watt rating) heating for 5 min at full power, resting for 5 min and reheated for 5 additional minutes. The citrate solution began to boil after 4 min in the first heating and boiled continuously during the second heating. Sections were left to cool in the citrate buffer for 30 min. Retrieved sections were rinsed in 50 mM Tris-buffered saline (TBS; pH 7.6), blocked for 30 min with 5% normal goat serum (NGS) in TBS, and incubated with either anti-Ki67 antibody (NCL-Ki67-MM1; Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) or BrdU monoclonal antibody (clone BMC 9318, Boehringer Mannheim) at 1 µg/mL in TBS 1% NGS overnight in a humidified chamber at 4°C. Slides were rinsed in TBS (3 x 5 min), exposed to unlabeled goat anti-mouse IgG diluted 1:200 in TBS 1% NGS plus 2% normal bovine serum for 30 min, and visualized by the peroxidase anti-peroxidase technique with Gill’s hematoxylin #2 diluted 1:1 with water (Garrett and Guthrie, 1998). Finally, slides were dehydrated in ascending alcohols and cleared in xylene, and glass coverslips were mounted with Permaslip. Negative control slides were prepared by substitution of mouse IgG for primary antibody (M9269, Sigma Immunochemicals, St. Louis, MO). Counter-stained sections were used to enumerate positive staining for Ki67 and BrdU incorporation and to determine villus height and crypt depth with magnification (1000 x under oil) using ocular reticles (B-0506 and B-0509; Optical Elements Corp., Dulles, VA). Percentage staining was determined by dividing the number of stained cells within a randomly selected field by total cells within the same field and multiplying by 100. For enumeration of Ki67 and BrdU positive staining cells, 10 villi were selected at random and mean percentage staining was recorded for each intestinal segment for each cow.

Statistical Analyses
All statistical analyses were conducted using GLM procedures of SAS (1990). Data were analyzed as a complete randomized design with the 4 stages of lactation as the main effect. Orthogonal polynomial contrasts were used to test for linear, quadratic, and cubic responses. All data are presented as least square means ± SE, with the SE calculated using the least number of observations for each measured variable. Treatment effects were considered significant at the P 0.05 level and a tendency to be significant at P < 0.10.

	RESULTS

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Lactational performance and mammary physiology was the focus of a concurrent aspect of this experiment and these data have been presented previously (Capuco et al., 2001). Daily milk yields were 39.9, 47.3, 44.6, and 36.5 kg/d for the 14, 90,120, and 240 DIM groups, respectively. Dry matter intake increased through 120 DIM and had declined by 240 DIM (quadratic; P = 0.0001; Table 2). Conversely, and as expected, both carcass and empty BW declined from early to peak lactation, but recovered by late lactation (quadratic; P 0.004). Similarly, live BW exhibited a characteristic decline followed by a recovery in late lactation (data presented in Capuco et al., 2001). Body condition mean scores were 2.97, 2.81, 2.40, and 3.21, respectively, for the 14, 90,120, and 240 DIM cows, reflecting the mobilization of body tissue stores in support of lactation and regeneration of body tissues during late lactation.

	DISCUSSION

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Organ weights reported by Smith and Baldwin (1974) for heart, lung, and spleen were greater in lactating cows than in nonlactating nonpregnant cows. The current experiment was not designed to make this same comparison. However, through lactation, the mass of heart, lung, and spleen tissues actually decreased on an absolute basis at 120 d of lactation from that at 14 d postpartum, following the pattern of the empty body mass of the cow. However, when expressed on an EBW basis, heart and spleen increased in mass with milk output. Surprisingly, lungs exhibited a slight decrease in mass with increasing DIM when corrected for EBW. Using a pregnant beef heifer model to ascertain the role of visceral organs to the increased maintenance energy requirement of pregnant animals, Scheaffer et al. (2001) were similarly unable to discern clear changes in service tissue masses. Consistent with Smith and Baldwin (1974) and Scheaffer et al. (2001), kidney weights in the current experiment did not follow a distinct pattern with nutritional demands imposed by lactation and pregnancy. Despite a 42% numerical increase in pancreatic tissue mass during lactation, statistical differences could not be discerned in the current experiment. Based on the reduction in visceral adipose tissue through 120 DIM, visceral adipose tissue pool was clearly mobilized to meet the metabolic demands of lactation and subsequently replenished when nutrient demands of the udder were presumably met by nutrients provided by the diet.

Due to their vital role in absorption and assimilation of nutrients and their relatively high metabolic activities, digestive tract tissues and liver are thought to influence maintenance energy requirements (Johnson et al., 1990). In ruminants, these organs are affected by changes in ME intake (Johnson et al., 1990; McLeod and Baldwin, 2000), protein intake (Wester et al., 1995; Baldwin et al., 2000), nutrient restriction and/or realimentation (Ferrell et al., 1986; Sainz and Bentley, 1997; Johnson et al., 1990) as well as energy density of the diet (Sainz and Bentley, 1995; McLeod and Baldwin, 2000). Changes in mass associated with physiological state, when dietary energy intake is maintained have been equivocal (Scheaffer et al., 2001). In the current experiment, small intestine weight and length, rumen mass, and liver mass exhibited disproportionate increases with the increased nutrient supply and demand occurring as a result of lactation. Interestingly, Sainz and Bentley (1995) demonstrated in growing steers that growth of the small intestine, liver, and fore stomachs were the result of different processes following realimentation (hyperplatic growth, hypertrophic growth, and both, respectively). Small intestinal growth responses generally appear to be due to increases in cell number across a variety of dietary treatments, including nutrient restriction (Sainz and Bentley, 1995; McLeod and Baldwin, 2000; Baldwin and McLeod, 2001; Swanson et al., 2001). In the current data set, increases in small intestinal mass were associated with greater DIM and DMI, while DNA concentrations were unaffected, suggesting that the increases in mass were due to increased cellularity. Further, using N:DNA ratio as an indicator of cell size (Enesco and Leblond, 1962) our data indicated that no cellular hypertrophy occurred for the small intestinal tissues.

Rumen epithelial growth in the current experiment also followed a pattern consistent with hyperplastic growth as tissue composition was unaffected by stage of lactation. This is different from reports in which ruminal growth has been attributed to both hyperplastic and hypertrophic growth. Growing lambs (McLeod and Baldwin, 2000) and finishing cattle (Sainz and Bentley, 1995) fed greater ME intakes exhibited increased rumen mass as a result of both hyperplastic and hypertrophic responses compared with animals fed at a lower ME intake. However, in restricted lambs (Burrin et al., 1992) decreased rumen mass was soley due to decreased cell numbers.

In the current experiment, change in liver mass, as a percentage of EBW, was due to both an increase in cell size and cell number, as both the N and DNA concentrations were affected by DIM and N:DNA was increased, with DIM indicating greater cell size. This is consistent with data from steers (Sainz and Bentley, 1995) and sheep (Swanson et al., 1999; McLeod and Baldwin, 2000) and consistent with the concept that liver size is related to the physiological workload and responds with increases in cell size to meet physiological demands (Johnson et al., 1990; Sainz and Bentley, 1995; McLeod and Baldwin, 2000). Although intestinal organ mass increased with increasing nutrient demand, DNA content of the organs was largely unaffected, consistent with an increase in tissue cellularity.

In this experiment we sought to determine if short-term measures of cellular proliferation would be useful for monitoring these apparent hyperplastic growth events. Although differences in BrdU labeling were apparent with stage of lactation, the pattern exhibited was not consistent with the growth responses observed. For instance, small intestinal mass increased to a maximum at 120 DIM due to increased cellularity mirroring the level of DMI and milk production, yet BrdU incorporation exhibited a cubic response with apparent peaks at 90 and 240 DIM. Swanson et al. (1999) used in vivo pulse labeling with BrdU to assess ewe intestinal growth in response to UIP and were unable to detect changes in labeling of cells. Thus, while the BrdU measurements are useful in delineating proliferative cells within tissues, and with appropriate methodology can provide true rates of cell proliferation, numerous tissue samples and a wide range of times may be needed to demonstrate the usefulness of this method as a predictor of increases in total tissue proliferation. For example, increased cell proliferation at 90 DIM contributes to tissue mass at this time point but can also contribute to increased tissue mass at 120 DIM. The duration of the increased proliferation was not assessed because of an insufficient number of time points. Furthermore, tissue sampling site and region within a tissue section chosen for counting are highly influential and, thus, acquiring representative samples is difficult. Although the Ki67 antigen staining protocol was not able to detect changes in proliferation, staining indicated that 6 to 8% rumen epithelial cells and 20 to 30% of the cells of the intestinal crypts are active in the cell cycle. Due to the large percentage of cells in the growth fraction, a small change in transit time through the cell cycle would elicit a large net effect on tissue proliferation. Thus, it is not necessarily contradictory to observe no change in Ki67 staining, while observing an increase in tissue cellularity. Tritiated thymidine incorporation by epithelial mucosal scrapings exhibited a response consistent with the observed increases in duodenal epithelial mass. This is consistent with a stable growth fraction (Ki67 measurement) with a decreased transit time through cell cycle as well as the observed whole tissue proliferation. However, because these measurements are made on a small portion of the whole tissue, slight variations are magnified when extrapolating to a whole tissue basis. For intestinal tissues, accurate measurement and prediction of sloughing of cells is not yet feasible. Thus, tissue samples must be carefully selected to ensure accurate representation of the whole tissue and interpretation must concomitantly consider whole tissue response.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Changes in visceral organ mass have long been implicated as a causative factor involved in the increase in maintenance energy requirements associated with lactation in dairy cattle. These data complement and extend earlier findings in dairy cattle by including measurement of tissue composition and illustrating that the growth phenomenon is associated with increased cellularity in the rumen and small intestine epithelia. Consistent with other physiological challenges that result in increased liver nutrient workload, increases in liver mass during lactation are primarily due to increasing cell size. Use of proliferation indices including, BrdU incorporation, Ki67 antigen staining, and tritiated thymidine incorporation assays present different methodological challenges for accurate assessment and prediction of tissue proliferation status in the lactating dairy cow. However, in conjunction with whole tissue measurements of proliferation, important insights into the dynamics of growth responses can be gained. Efforts to accurately predict maintenance requirements and/or modify the efficiency of milk production need to consider the dynamics of visceral tissue mass in response to stage of the lactation cycle.

	FOOTNOTES

 
^* The authors gratefully acknowledge D. Hucht, M. Miner, M. Niland, J. Wilson, and L. Wood for technical assistance, R. Zephir and J. Piatt for abattoir assistance.

Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by USDA and does not imply its approval to the exclusion of other products that may be suitable.

Received for publication December 27, 2002. Accepted for publication August 18, 2003.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 


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