Abomasal Infusion of Glucose and Fat Effect on Digestion, Production, and Ovarian and Uterine Functions of Cows 1

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1 Abomasal Infusion of Glucose and Fat Effect on Digestion, Production, and Ovarian and Uterine Functions of Cows 1 B. S. OLDICK,*,2 C. R. STAPLES,*,3 W. W. THATCHER,* and P. GYAWU *Department of Dairy and Poultry Sciences, University of Florida, Gainesville University of Science and Technology, Kumasi, Ghana ABSTRACT Four ruminally cannulated lactating dairy cows, arranged in a 4 4 Latin square design, were infused abomasally with 1) water (control), 2) 1 kg/d of glucose, 3) 0.45 kg/d of tallow, and 4) 0.45 kg/d of yellow grease. Cows were synchronized for estrus within each 35-d period by injection of a GnRH agonist followed 7 d later by an injection of PGF 2a. Dry matter intake was not affected by infusates. Apparent digestibility of total fatty acids was greater for cows receiving the fat infusions relative to those receiving the glucose infusion and tended to increase for cows receiving the yellow grease infusion compared with those receiving the tallow infusion. Energy infusions decreased apparent acid detergent fiber digestibility compared with effects of the control infusion. Fat infusions tended to increase milk fat percentage and decrease the energy status of cows relative to the glucose infusion. The feed efficiency was greater for cows receiving fat infusions than for those receiving the glucose infusion and was greater for cows receiving the yellow grease infusion than for those receiving the tallow infusion. Plasma progesterone concentration peaked higher during the estrous cycle for cows infused with fat than for those infused with glucose. Mean growth rate and maximum size of the first wave dominant follicle were greater with tallow than with yellow grease. During the period of infusion of yellow grease and afterward, release of 13,14-dihydro-15-keto-PGF 2a in response to an injection of oxytocin on d 15 of the estrous cycle was attenuated. ( Key words: abomasum, fat, digestion, reproduction) Received October 16, Accepted October 11, Florida Agricultural Experiment Station Journal Series Number R Present address: Department of Animal Sciences, The Ohio State University, Columbus Reprint requests. Abbreviation key: CL = corpus luteum, PES = prostaglandin endoperoxide synthase, PGFM = 13,14-dihydro-15-keto-PGF 2a. INTRODUCTION As the energy demands of cows in early lactation increase because of increased milk production and as sources of animal fat become more readily available and less expensive, the use of fats in the diets of dairy cows is likely to increase. Tallow and yellow grease are fat sources that are readily available. Yellow grease is generally more unsaturated than tallow and contains more linoleic acid, an essential fatty acid, than does tallow (12, 35). The degree of saturation of supplemental dietary fat influences digestion and absorption of the fat (3, 23). More saturated fat generally has fewer detrimental effects on microbial metabolism in the rumen, and more unsaturated fat is absorbed more effectively in the small intestine. The type of supplemental fat provided may also influence production responses. Milk production was often increased when diets were supplemented with yellow grease (5, 12). However, production of milk or FCM was not affected when diets based on alfalfa silage and corn silage were supplemented with tallow (19). Only when alfalfa hay was fed with corn silage (39) and when tallow was hydrogenated (14) was milk production increased. Addition of fat to diets could potentially increase net energy intake and thus decrease the duration and magnitude of negative energy status. In general, lactating cows in severe negative energy balance have poor reproductive performance (4). Dietary fat also may serve as a precursor for the synthesis of progesterone (via increased cholesterol release and synthesis) and prostaglandins (from supply of linoleic and arachidonic acids); both have been linked to improved fertility of dairy cows (15, 27). Therefore, dietary fat may affect reproductive performance independently of the effects on energy status. Reproductive performance was improved for postpartum cows fed supplemental fat, although supplemen J Dairy Sci 80:

2 1316 OLDICK ET AL. tal fat caused greater BW loss (37) and lower energy status (38). Research is lacking that independently considers the effects of energy status, energy source, and fatty acid profiles of supplemental fat on reproduction. The objective of this study was to examine the influences of changing energy status, supplemental energy source (carbohydrate vs. fat), and supplemental fatty acid profiles in the diets of lactating dairy cows on the dynamics of ovarian follicles, uterine synthesis of PGF 2a, plasma hormone and metabolite profiles, milk production and composition, and feed intake and digestibility. MATERIALS AND METHODS Cows, Diet, and Facilities Four ruminally cannulated multiparous Holstein cows (mean DIM, 111 ± 14) were arranged in a 4 4 Latin square design. Cows had demonstrated estrous behavior before initiation of the experiment. Each period lasted 35 ± 2 d; 14 d were allowed for adjustment to treatments, and 21 d were allowed for data collection. Cows were housed in tie stalls within the same barn at the University of Florida Dairy Research Unit (Hague). The experiment took place from February 16 to July 18, Evaporative cooling and 1.2-m box fans were used when needed to maintain a temperature of 30 C in the barn. Cows were released into a dirt exercise lot from 0000 to 0700 h each day. Cows were fed a TMR twice daily at approximately 1000 and 1600 h in amounts to ensure 5% orts. Orts were weighed and discarded just before the feeding at 1000 h. The TMR were formulated to contain (DM basis) 25.8% corn silage, 11.2% cottonseed hulls, and 63.0% concentrate to meet nutrient recommendations (30) (Table 1). Cows were milked three times daily at 0700, 1500, and 2300 h. Milk production was recorded at each milking. Treatments The four treatments consisted of abomasal infusions of 1) water (control), 2) 1 kg/d of glucose, 3) 0.45 kg/d of tallow, and 4) 0.45 kg/d of yellow grease. To maintain emulsions that were suitable for infusion, the fat sources were mixed with meat solubles (Milk Specialties Co., Dundee, IL). To standardize treatments, equal amounts of meat solubles were added to each infusate. Meat solubles (168 g) were dissolved in 1 L of hot (50 to 60 C) tap water. For the water and glucose infusions, this solution was added to a 20-L Nalgene container (Fisher Scientific, Pittsburgh, PA) and placed on a stir plate. Tap water was TABLE 1. Ingredient and chemical concentration of the experimental diet fed to lactating Holstein cows abomasally infused with water, glucose, tallow, and yellow grease. Composition Ingredient, % of DM Corn silage 25.8 Ground corn 26.6 Cottonseed hulls 11.2 Distillers dried grains with solubles 11.2 Blood meal 1.6 Soybean meal 6.6 Whole cottonseeds 11.2 Mineral mix Total Chemical DM, % 58.6 OM, % of DM 93.0 NE L, Mcal/kg of DM 1.61 CP, % of DM 20.1 ADF, % of DM 24.1 NDF, % of DM 36.7 Fatty acids, % of DM 5.2 Ca, % of DM 1.08 P, % of DM 0.54 Mg, % of DM 0.36 K, % of DM 1.10 Na, % of DM 0.54 S, % of DM 0.27 Fe, ppm in DM 279 Zn, ppm in DM 79 Cu, ppm in DM 25 Mn, ppm in DM 64 Mo, ppm in DM 1 1Contained 12.0% Ca, 2.8% P, 2.9% Mg, 1.0% S, 1.6% K, 7.8% Na, 800 ppm of Zn, 320 ppm of Cu, 800 ppm of Fe, 800 ppm of Mn, 242,000 IU of vitamin A/kg, 35,200 IU of vitamin D/kg, and 880 IU of vitamin E/kg. Soybean hulls served as a carrier. added to the water infusate to bring the final volume to 11 L. The solution was stirred constantly using a 10-cm stir bar. To prepare the glucose infusate, 1 kg of glucose powder (Fisher Scientific) was added slowly to 2 L of hot (50 to 60 C) tap water. After the solution was transferred to a 20-L Nalgene container, tap water was added to bring the final volume to 11 L. The solution was stirred constantly using a 10-cm stir bar. Each fat infusate was prepared in two equal volumes. Tallow (Griffen Industries, Inc., Cold Spring, KY) and yellow grease (Griffen Industries, Inc.) were heated in a conventional oven at 150 C for approximately 5 min. Liquefied fat sources (0.23 kg per flask) were added slowly to 6-L Pyrex flasks (Fisher Scientific) containing the meat solubles solution. Hot (50 to 60 C) tap water was added to the flasks to bring the final volume to 5.5 L. Temperature was maintained at 60 C using a heating stir plate, and the solution was stirred constantly. Infusion of the contents of one flask (5.5 L) began at 0800 h, and infusion of the contents of the second flask (5.5 L)

3 FAT INTAKE AND OVARIAN AND UTERINE FUNCTION 1317 began at approximately 1600 h. The temperature of infusate entering the rumen was approximately 40 C. Approximately equal volumes (11 L) of the emulsions and solutions were infused continuously (11.5 ml/min) over a 16-h period (0800 to 2400 h) each day using peristaltic pumps (for fat infusates, Harvard Apparatus model 1210; Harvard Apparatus, Inc., South Natick, MA; for glucose and water infusates, Haake Buchler Multistatic Pump model ; Haake Buchler Instruments, Inc., Saddle Brook, NJ). The infusion apparatus consisted of a glass tube (0.48 cm i.d.; 0.64 cm o.d.) that was submerged in the emulsion and solution and was connected to tygon tubing (0.48 cm i.d.; 0.64 cm o.d.). For the glucose and water infusions, tygon tubing (0.48 cm i.d.; 0.64 cm o.d.) carried the infusate to the side of the cow where a 3- to 6-mm reducing connector (Fisher Scientific) joined the tubing to tygon tubing with a thicker wall (0.32 cm i.d.; 0.64 cm o.d.). The tygon tubing carried the infusate through the ruminal fistula and sulcus omasi to the abomasum where a perforated 60-ml polypropylene bottle was attached to the end of the tubing. The bottle was anchored into the abomasum with a circular flange that was 13 cm in diameter. The flange was located approximately 10 cm from the bottle. The bottle and the circular flange, which were connected to the tubing, were squeezed together in the left hand. The hand was inserted through the ruminal fistula and through the sulcus omasi. Moving downward and caudally, the abomasum was entered as was evident by the feel of slick tissue. Then, the flange was unfolded, which prevented the exit of the bottle. For the fat infusions, tygon tubing (0.32 cm i.d.; 0.64 cm o.d.) was connected to larger tubing (0.48 cm i.d.; 0.64 cm o.d.) near the glass rod using a 3- to 6-mm reducing tubing connector. The smaller diameter tygon tubing carried the infusate to a perforated 60-ml polypropylene bottle that was anchored in the abomasum as was previously described for the water and glucose infusates. For the yellow grease infusion, electrical heating tape and insulation were wrapped around the infusion tubing from the infusion pump to the side of the cow to prevent the infusate from solidifying in the tubing. For the tallow infusion, a water bath circulated hot (approximately 50 C) water through a tygon hose, which ran parallel to the tallow infusion line from the infusion pump to the side of the cow. The tallow infusion line and the water hose were wrapped together with insulation. Placement of the infusion lines was checked several times during each period by inserting a hand into the abomasum to ensure that the infusate was being delivered into the abomasum. At no time was the bottle found outside the abomasum. The bottle was removed at the conclusion of each period, cleaned, and reinserted at the start of the next period. Reproductive Management Estrous cycles were synchronized at the beginning of each period. A GnRH agonist (Buserelin; Hoechst- Roussel Agri-Vet Co., Somerville, NJ) was administered (8 mg) on d 10 of each period, followed by an injection of PGF 2a (25 mg; Lutalyse ; The Upjohn Co., Kalamazoo, MI) on d 3 of each period. Day 1 was defined as the day of ovulation. Cows that did not respond to PGF 2a with regression of the corpus luteum ( CL) (as determined using ultrasonography) were given a second injection of PGF 2a. On d 15 of the following synchronized estrous cycle, an oxytocin challenge (100 U) was administered intravenously via a jugular catheter (Tech America, Fermenta, Kansas City, MO). On the day following the oxytocin challenge, PGF 2a (25 mg) was administered to regress the CL and to induce terminal development of an ovulatory follicle from the second wave dominant follicle. Ovarian Follicular Dynamics Ultrasound procedures (26) were used to follow the follicular dynamics of synchronized estrous cycles. Cows were examined by transrectal ultrasonography (Equisonics 300A linear-array ultrasound scanner; Tokyo Keiki Co. Ltd., Tokyo, Japan) from the day of initial PGF 2a injection (d 3) to the day of ovulation following oxytocin challenge. This ovulation marked the end of each experimental period for each cow. The size and relative position of ovarian follicles that were >3 mm in diameter and the presence of other ovarian structures (e.g., CL) were mapped daily. The size and number of ovarian structures were determined by retrospective examination of ovarian maps. The day of CL emergence was defined as the first day of the estrous cycle at which time the CL was visible by ultrasonography. The day of CL disappearance was defined as the first day of the estrous cycle at which time the CL was no longer visible by ultrasonography. The day of the emergence of dominant follicles was defined as the day that the dominant follicle was at least 2 mm larger than the subordinate follicle. The day that the first dominant follicle stopped growing was estimated by regression analysis. The diameters of first dominant follicles were fit to second-order regression curves within cow and period. The first derivative of the resulting regression equation was determined and set equal to zero. Solving this equation yielded an estimate of the

4 1318 OLDICK ET AL. day on which the dominant follicle of the first wave experienced no growth. The growth rate of the dominant follicle of the first wave was defined as the slope of the linear regression line of the follicle diameter over time from the day of emergence to the day that the follicle experienced no growth. The day that the first dominant follicle lost dominance was defined as the day on which the number of 6- to 9-mm follicles increased. The growth rate of the second dominant follicle was defined as the slope of the linear regression line of the diameter of the second dominant follicle over time from the day of emergence to the day of ovulation. Sampling and Analyses Daily DMI (feed offered minus orts) and milk production were recorded for each cow from d 0 to 18 of each synchronized estrous cycle (approximately d 15 to 33 of each period). Feed samples were collected weekly, dried at 55 C for 48 h, composited by period, and analyzed for CP, NDF, ADF, Ca, P, Mg, K, Na, S, Zn, Cu, Mn, Mo, Fe (Northeast DHIA Forage Laboratory, Ithaca, NY), ash (1), and fatty acids (42). Concentration of dietary NE L was calculated using NRC (30) values for individual feedstuffs. Milk was sampled from six consecutive milkings during wk 4 and 5 of each period and analyzed for fat and protein (Southeast Dairy Laboratory, McDonough, GA). Daily blood samples were taken via the jugular or coccygeal vein from d 0 to ovulation at approximately 1000 h. All blood samples were collected in sodium heparinized tubes, immediately placed on ice, and centrifuged at 3000 g at 4 C for 15 min. Plasma was stored at 20 C for later analyses. Plasma samples taken from d 0 to 18 were analyzed for insulin (10), triglycerides (16), cholesterol (kit no. 352; Sigma Chemical Co., St. Louis, MO), NEFA (modified NEFA-C procedure; WAKO Chemicals USA, Inc., Richmond, VA), and glucose (kit no. 315; Sigma Chemical Co.). All plasma samples were analyzed for progesterone (24) and estradiol (2). On d 15 of each synchronized estrous cycle, cows were injected intravenously with oxytocin (100 U) to stimulate the release of PGF 2a from uterine tissues (oxytocin challenge). Blood samples were taken via indwelling jugular catheters at 60, 45, 30, 15, 0, 5, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, and 240 min relative to the time of the oxytocin injection. Indwelling jugular catheters were inserted 4 h prior to oxytocin injection. Plasma was harvested, stored as was described previously, and later assayed for 13,14,-dihydro-15-keto-PGF 2a ( PGFM) (20). Repeated measurements of two plasma sample pools across several assays were used to calculate intraassay and interassay coefficients of variation for each hormone and metabolite measured. Apparent Digestibility Apparent digestibilities of DM, OM, CP, NDF, ADF, and fatty acids were measured using the marker ratio technique (36). Cows were dosed via the ruminal fistula with gelatin capsules containing 10 g of Cr 2 O 3. Dosing occurred at 1100 and 2300 h for 10 d. During the last 5 d, fecal grab samples were collected at the time of dosing and stored at 20 C. Upon thawing, fecal samples were composited on a wet basis, dried at 55 C, and analyzed for DM, OM, ADF, CP (1), and NDF (44) using a heat-stable a-amylase enzyme (A-3306; Sigma Chemical Co.) and fatty acids (42). Energy Status Daily energy status was calculated using the following equation (30): energy status = net energy intake net energy for maintenance NE L, where net energy intake (megacalories per day) = DMI (kilograms per day) net energy density of feed (megacalories per kilogram), net energy for maintenance (megacalories per day) = BW (kilograms) megacalories per kilogram of BW per day, and NE L (megacalories per day) = milk production (kilograms per day) [ ( percentage of milk fat)]. Energy density of feed was determined from the percentage of OM digestibility using the following equation: energy density of feed (megacalories per kilogram) = OM digestibility (29). Body weight was determined once each week at 0700 h. Statistical Analyses Data were analyzed by least squares ANOVA using the general linear models procedure of SAS (34). For analyses of plasma metabolite and hormone concentrations the model was Y ijkl = m +T i +C j +P k + TCP ijk +D l +DT li +DC lj +DP lk +e ijkl where m = overall population mean, T i = effect of infusion treatment, C j = effect of cow, P k = effect of experimental period, TCP ijk = effect of treatment by cow by experimental period interaction, D l = effect of day, DT li = effect of day by treatment interaction, DC lj = effect of day by cow interaction, DP lk = effect of day by period interaction, and e ijkl = residual error term. The error term, TCP ijk, was used to test for main effects of treatment, cow, and period. The residual

5 FAT INTAKE AND OVARIAN AND UTERINE FUNCTION 1319 was used as the error term to test for main effects of day and interactions with day. Intake and production variables were reduced to means for each treatment for each cow for each period before statistical analyses. For analyses of these means, plus energy status, BW change, apparent digestibilities, and ovarian measurements, the model was Y ijk = m +T i +C j +P k +e ijk where m = overall population mean, T i = effect of infusion treatment, C j = effect of cow, P k = effect of experimental period, and e ijk = residual error term. The residual was used as the error term to test for main effects of treatment, cow, and period. During period 1, the cow receiving the tallow infusion developed a follicular cyst. Reproductive hormone and ovarian data from this period for this cow were deleted prior to statistical analyses. Tests of homogeneity of regression (47) were performed on plasma concentrations of reproductive hormones. Polynomial regression curves were fit to each independent variable versus day to describe a single pooled curve across all treatments. The F test involved the gain from fitting individual regression curves for treatments compared with fitting the single pooled treatment curve. A similar test of homogeneity of regression was performed on plasma PGFM concentration over 240 min following oxytocin challenge. Prior to this analysis, observations were divided into two groups because of a suspected residual effect. The groups (treatments) were 1) yellow grease (cells in which yellow grease was the infusate or had been the infusate previously) and 2) no yellow grease (all other cells of data). Orthogonal contrasts for all variables except plasma PGFM concentrations were 1) water versus glucose plus tallow plus yellow grease, 2) glucose versus tallow plus yellow grease, and 3) tallow versus yellow grease. Contrast 1 examined the effect of energy supplementation, contrast 2 examined the effect of energy source (e.g., carbohydrate vs. fats), and contrast 3 examined the effect of different fatty acid profiles. Significance was declared at P < 0.05, and a tendency toward significance was declared at 0.05 < P < RESULTS AND DISCUSSION TABLE 2. Fatty acid (FA) profiles of the experimental diet, tallow, and yellow grease and other characteristics of tallow and yellow grease given to lactating Holstein cows. Item Diet Tallow Yellow grease (% of total FA) FA C 13: C 14: C 14: C 15: C 16: C 16: C 17: C 18: trans-c 18: cis-c 18: C 18: C 18: C 20: C 20: C 20: C 22: C 24: C 22: Other Total FA, % Saturated FA, % Unsaturated FA, % Free FA, 1 % Moisture, 1 % Impurities, 1 % Iodine value As provided by Griffen Industries, Inc. (Cold Spring, KY). Diet and Composition of Fat Source Ingredients and the chemical composition of diets are shown in Table 1. Diets were formulated to meet NRC (30) recommendations. The dietary concentration of RUP, calculated from NRC (30) values for individual feed ingredients, was 35% of total CP. Fatty acid composition of the experimental diet (Table 2) consisted of primarily C 16:0, cis-c 18:1, and C 18:2. However, the majority of dietary unsaturated fatty acids are biohydrogenated in the rumen, and C 18:0 is the primary dietary fatty acid that passes to the duodenum. The yellow grease and tallow contained 77.3 and 83.1% fatty acids (DM basis), respectively (Table 2). Yellow grease contained a greater proportion of C 18:2 (17.36% vs. 2.09%) and smaller proportions of C 16:0 (16.60% vs %) and C 18:0 (9.59% vs %) relative to tallow. The fat sources contained similar proportions of C 18:1 and C 18:3, which can inhibit conversion of C 18:2 to C 20:4 (21). These fatty acids, C 18:1 and C 18:3, also can inhibit cyclooxygenase activity and subsequent prostaglandin synthesis in the vesicular gland tissue of sheep (25). Both fat sources were low in impurities. The fatty acid content of commercially available tallow and yellow grease can be quite variable. Effects on animal performance may be dependent on the fatty acid profile of the fat source; therefore,

6 1320 OLDICK ET AL. sources of commercial tallow with different fatty acid profiles may exert different effects. Nutrient Intake and Apparent Digestibilities Mean intakes and infusions of DM (kilograms per day and percentage of BW), OM, NE L, CP, ADF, and NDF (Table 3) were not affected by infusate treatments. In previous studies (9, 45), abomasal infusion of glucose did not affect DMI. Similarly, in the present study, approximately 450 ml/d of yellow grease and tallow were infused into the abomasum without affecting DMI. Postruminal infusions of triglycerides at 500 ml/d (33) and 900 ml/d (18) have previously depressed DMI. Christensen et al. ( 8 ) and Drackley et al. (13) reported that DMI was decreased when 0.45 kg/d of free fatty acids were infused postruminally into lactating dairy cows. The DMI decreased linearly as the degree of unsaturation and carbon chain length of the infused free fatty acids increased (13). However, DMI was not depressed when 1.5 kg/d of rapeseed oil were infused into the duodenum of cows in late lactation (7). Mechanisms by which fats affect DMI, especially postruminally, require further study. Cows infused with fat received approximately 400 g/d more fatty acids ( P = 0.001) than did cows infused with water or glucose (Table 3). Amounts of specific fatty acids that differed the most between tallow and yellow grease infusates were, respectively: C 16:0 (95 vs. 58 g/d), C 18:0 (84 vs. 33 g/d), trans-c 18:1 (14 vs. 34 g/d), and C 18:2 (8 vs. 60 g/d). Apparent digestibilities of DM, OM, and CP in the total tract were not affected by infusates, indicating that the treatments did not compromise ruminal and postruminal digestion of these constituents (Table 3). Others have reported no change in the total tract apparent digestibility of DM by lactating cows that were abomasally infused with glucose ( 9 ) or fat (7, 8, 13). In previous studies (8, 13), postruminal infusion of mostly unsaturated fat sources decreased digestibility of CP compared with more saturated fat sources. McCarthy et al. (28) reported that 28% of apparent total tract digestion of NDF occurred postruminally in lactating dairy cows, indicating the importance of postruminal fiber digestion. Total tract apparent digestibility of ADF was decreased when energy (glucose, tallow, or yellow grease) substrates were infused abomasally relative to the water infusion (Table 3). Infusion of yellow grease tended to depress digestion of NDF compared with the infusion of tallow. This depression likely occurred during microbial digestion in the cecum. Apparent digestibility of NDF was not affected by abomasal infusions of saturated or unsaturated fats in other studies (8, 13) and was not affected when mostly saturated fats were fed (14). However, supplemental yellow grease in the diet often decreased apparent NDF digestibility (5). TABLE 3. Mean intake (including amount of energy infused) and apparent digestibilities of DM, OM, CP, ADF, and NDF by lactating Holstein cows abomasally infused with water (W), glucose (G), tallow (T), and yellow grease (YG). Orthogonal contrast Infusate W vs. (G G vs. Item W G T YG SE +T+YG) (T + YG) T vs. YG P Intake DM, kg/d DM, % of BW OM, kg/d NE L, Mcal/d CP, kg/d ADF, kg/d NDF, kg/d Fatty acids, kg/d (%) Apparent digestibility DM OM CP ADF NDF

7 FAT INTAKE AND OVARIAN AND UTERINE FUNCTION 1321 Because dietary intake, but not abomasal infusion, of yellow grease has affected NDF digestion, the negative effects of unsaturated fats on NDF digestion probably were associated mainly with ruminal microorganisms rather than with cecal microorganisms. Increased concentrations of unsaturated fatty acids, such as those in yellow grease, may upset the cellular membrane structure and function of ruminal microorganisms (D. B. Bates, 1996, personal communication). Apparent digestibility of total fatty acids increased in cows receiving fat infusions (Table 4). Highly digestible supplemental fat may dilute endogenous fatty acids from bile salts or bacteria (3). Fatty acids with the greatest digestibility were C 14:0,C 16:1,C 18:0, and C 20:0. Nearly 100% of linoleic and linolenic acids in tallow and yellow grease were digested. Apparent digestibility of fatty acids was decreased (14) or did not change (35) in response to supplemental tallow. Apparent digestibility of fatty acids tended to be greater when yellow grease was infused than when tallow was infused (Table 4). This result supports the hypothesis that, relative to unsaturated fatty acids, saturated fatty acids are absorbed less in the small intestine (3). Unsaturated fat sources were more digestible than saturated fat sources in a previous study (13). Milk Production and Composition Mean milk production was not affected by treatments, although mean 4% FCM production tended to be greater when fats were infused than when glucose was infused (Table 5). Previously, production of milk (8, 13) and FCM ( 8 ) were decreased when 0.45 kg/d of free fatty acids were infused abomasally in high producing dairy cows. In several other studies (7, 18, 33), however, milk production was not affected by abomasal infusion of fat. When compared with the infusion of mainly saturated fat sources, infusion of mainly unsaturated fats did not affect milk production (8, 13). Cows infused with fat produced milk and 4% FCM more efficiently than did those receiving the glucose infusion (Table 5). When fat was infused, direct incorporation of long-chain fatty acids into milk might have decreased de novo fatty acid synthesis by the mammary gland and, subsequently, increased efficiency of milk synthesis. Cows infused with yellow grease did experience increased efficiencies of milk production compared with cows infused with tallow. This increase was likely the result of a nonsignificant decrease in DMI when yellow grease was infused relative to the infusion of tallow (21.5 vs kg/d) without any subsequent effect on production of milk. In addition, greater digestion of total fatty acids by cows infused with yellow grease might have improved efficiency of milk production. Milk protein concentration remained stable across infusion treatments with one exception. Milk protein percentage was lower when yellow grease was infused than when tallow was infused (Table 5). In previous studies, postruminal infusion of fat has resulted in both decreased ( 8 ) and unchanged (13, 18) milk protein concentrations. The delivery of fat postruminally suggests that factors unrelated to the rumen were involved in milk protein depression. Although not investigated in the present study, possible mechanisms causing milk protein depression include decreased amino acid uptake by the small intestine, TABLE 4. Mean apparent digestibilities of fatty acids (FA) by lactating Holstein cows abomasally infused with water (W), glucose (G), tallow (T), and yellow grease (YG). Orthogonal contrast Infusate W vs. (G G vs. FA W G T YG SE +T+YG) (T + YG) T vs. YG (%) P C 13: C 14: C 16: C 16: C 18: cis-c 18: C 18: C 18: C 20: C 22: C 24: Other Total

8 1322 OLDICK ET AL. TABLE 5. Effect of abomasal infusion of water (W), glucose (G), tallow (T), and yellow grease (YG) on milk production, milk production efficiency, milk composition, energy status, and BW change of lactating Holstein cows. 1Milk production/(dmi + infusion) 100%. 24% FCM Production/(DMI + infusion) 100%. Orthogonal contrast Infusate W vs. (G G vs. FA W G T YG SE +T+YG) (T + YG) T vs. YG P Milk production, kg/d % FCM Production, kg/d Milk production efficiency, 1 % % FCM Production efficiency, 2 % Milk protein, % Milk fat, % Protein production, kg/d Fat production, kg/d Energy status, Mcal/d BW Change, kg/d decreased mammary blood flow (6), and insulin resistance (32), each of which could potentially decrease uptake of amino acids by the mammary gland. However, efficiency of amino acid transport by the mammary gland increased when fat was fed (11), suggesting that extraction of amino acids was not depressed. Infusates did not affect protein production measured in kilograms per day. Mean milk fat percentage and production tended to increase when fat was infused relative to glucose infusion. In previous studies, milk fat concentration was decreased (45) or left unaffected ( 9 ) by abomasal infusions of glucose. Infusion of highly unsaturated (91.6%) (17) or saturated (85.6%) (13) fat sources increased milk fat content of dairy cows. The saturated fatty acid content of yellow grease and tallow was 28.4 and 52.8%, respectively (Table 2). However, infusion of fat sources of intermediate saturation (54.2%) have decreased milk fat content (13). Energy Status and BW Change All cows were in positive energy balance in each period. Mean daily energy status tended to decrease when fat was infused relative to the energy status when cows were infused with glucose (Table 5). This decrease likely was because cows that were infused with fat tended to produce more 4% FCM than did cows infused with glucose; DMI was unchanged. Decreased energy status did not signify lower mean daily BW change, and BW change was unaffected by infusates. Blood Constituents Cows that were abomasally infused with fat experienced increased plasma concentrations of NEFA (Table 6). Similarly, plasma NEFA concentrations were increased by postruminal fat infusions in previous studies (13, 17, 33). The increase in plasma TABLE 6. Effect of abomasal infusion of water (W), glucose (G), tallow (T), and yellow grease (YG) on plasma constituents of lactating Holstein cows. 1Intraassay and interassay coefficients of variation were, respectively, 4.6 and 6.5% for NEFA, 4.6 and 8.5% for triglycerides, 2.1 and 2.0% for cholesterol, 1.2 and 2.4% for glucose, and 6.6 and 3.2% for insulin. Orthogonal contrast Infusate W vs. (G G vs. Constituent 1 W G T YG SE +T+YG) (T + YG) T vs. YG P NEFA, meq/l Triglyceride, mg/dl Cholesterol, mg/dl Glucose, mg/dl Insulin, ng/ml

9 FAT INTAKE AND OVARIAN AND UTERINE FUNCTION 1323 NEFA concentration might have been due to increased mobilization of adipose tissue or to inefficient uptake of fatty acids by cells after triglycerides were hydrolyzed by lipoprotein lipase activity (17). All cows in this study were in positive energy balance and were gaining BW. Therefore, cows receiving fat infusions were not likely to have mobilized large amounts of fatty acids from adipose tissue. Cows receiving abomasal infusions of glucose exhibited the lowest plasma NEFA concentrations. Increased plasma insulin concentrations that resulted when glucose was infused (Table 6) suppressed mobilization of fatty acids and, therefore, decreased plasma NEFA concentrations. Plasma triglyceride concentrations were greater for cows infused with fats than for those infused with glucose (Table 6), which suggested that abomasal infusion of fat increased the absorption of fat. Traditional diets are not likely to saturate the capacity of the small intestine to absorb fatty acids. Delivery of 1600 g of fatty acids to the lower gut (as occurred in this study) resulted in high (75%) rates of absorption (41). However, the true digestibility of fatty acids appeared to be decreased from 100 to 78% as fat intake increased from 1 to 8% of dietary DM (31). Cows that were infused with fat had greater plasma cholesterol concentrations than did those infused with glucose (Table 6). Similar results have been reported elsewhere when fat was infused postruminally (13, 17), although Christensen et al. ( 8 ) and Rindsig and Schultz (33) reported that plasma cholesterol concentrations were not affected by fat infusion. As expected, plasma concentrations of glucose and insulin were greater in cows receiving the glucose infusion than in those receiving fat infusion (Table 6). An increase in plasma glucose concentration without any subsequent increase in milk production (Table 5) suggests a limited capacity for glucose uptake by the mammary gland. The pool of follicles from which the first wave dominant follicle was recruited was composed of class 2 follicles. As the dominant follicle developed, plasma concentrations of estradiol increased slightly (Figure 1A). Increased plasma concentrations of progesterone promoted follicle turnover and suppressed continued increases in plasma estradiol concentrations. The first wave dominant follicle lost dominance as the plasma concentration of progesterone peaked (approximately d 12). With the loss of dominance, the number of class 2 follicles again increased (Figure 1B). A second wave dominant follicle emerged from the pool of class 2 follicles (Figure 1A). Injection of PGF 2a on d 15 regressed the CL, and plasma concentrations of progesterone declined. With decreased progesterone concentrations, the second dominant follicle was able to continue to grow. As the second dominant follicle Reproductive Hormones During the estrous cycle of each experimental period, plasma concentrations of progesterone and estradiol, as well as growth of ovarian follicles, followed a predictable profile (Figure 1). Intraassay and interassay coefficients of variation for progesterone were 6.7 and 10.7% and, for estradiol, were 11.3 and 1.0%, respectively. Following ovulation, plasma concentrations of progesterone increased as the CL developed. During these early days, the number of class 2 follicles (diameter, 6 to 9 mm) increased (Figure 1B). Figure 1. Panel A represents the mean (across treatments) plasma progesterone ( o) and estradiol ( ÿ) concentrations and the diameters of first ( ) and second ( π) wave dominant follicles for lactating Holstein cows during a synchronized estrous cycle. Standard errors of the means for progesterone, estradiol, first wave dominant follicles, and second wave dominant follicles were 0.3 ng/ ml, 0.2 pg/ml, 0.2 mm, and 0.2 mm, respectively. Panel B represents the mean number (across treatments) of class 1 (diameter, 5 mm; o), class 2 (diameter, 6 to 9 mm; ÿ), and class 3 (diameter, 10 mm; ) follicles for lactating Holstein cows during a synchronized estrous cycle. Standard errors of the means for number of class 1, class 2, and class 3 follicles were 0.4, 0.1, and 0.1, respectively.

10 1324 OLDICK ET AL. grew, estradiol concentrations increased. Estradiol acted positively on the hypothalamus and pituitary gland to induce a preovulatory surge of luteinizing hormone, resulting in ovulation by the second wave dominant follicle (approximately d 20). Mean plasma progesterone concentrations during a synchronized estrous cycle were not affected by treatments (overall mean, 5.29 ± 0.38 ng/ml). However, when curves of plasma progesterone concentrations over time were compared, treatment effects were detected. From d 0 to 15 of synchronized estrous cycles, the rate of increase in plasma progesterone concentration was greater in cows infused with water than when values were pooled across glucose, tallow, and yellow grease infusates combined (Figure 2A). Polynomial regression over time indicated also that progesterone concentration peaked higher for cows receiving the control infusion than for cows receiving energy infusions (12.3 vs ng/ml at d 13). The lower peak concentration of progesterone that occurred when energy sources were infused was primarily due to glucose infusion. Different polynomial regression curves ( P < 0.01) of plasma progesterone concentration over time indicated that progesterone peaked higher for cows receiving fat infusions than for cows receiving the glucose infusion (11.7 vs. 9.3 ng/ml at d 12.5) (Figure 2B). Increased amounts of dietary fat have been shown to increase plasma progesterone concentrations during certain portions of the estrous cycle (37). Increased progesterone concentrations that result when fat intake is increased may be caused by increased substrate (cholesterol) availability, although a direct link has not been established. Decreased plasma concentrations of progesterone have been associated with compromised reproductive performance (15). Treatment differences among progesterone curves were not detected during CL regression from d 16 to 20. The type of fat infused (tallow vs. yellow grease) did not influence plasma progesterone concentration when effects were evaluated using homogeneity of regression procedures. Mean plasma estradiol concentrations from d 1 to ovulation of a synchronized estrous cycle were decreased when energy sources were infused (2.43 vs ± 0.13 pg/ml; P = 0.042), primarily because of the effect of fat infusions ( P = for glucose vs. tallow plus yellow grease; 2.30 vs ± 0.13 pg/ml). These differences were mainly due to concentrations during d 15 to 20 of the estrous cycle (proestrus phase; Figure 2). Infusion of energy relative to control infusion (2.86 vs ± 0.34 pg/ml; P = 0.045) Figure 2. Mean plasma progesterone and estradiol concentrations for lactating Holstein cows infused with water (W), glucose (G), tallow (T), and yellow grease (YG) during a synchronized estrous cycle. Panel A represents concentrations for cows infused with W ( o) versus those for cows infused with G plus T plus YG ( ÿ) ( P < 0.01). The standard errors of the means for progesterone were 0.26 and 0.15 ng/ml, and the standard errors of the means for estradiol were 0.3 and 0.2 pg/ml, for the curves representing the W infusate and the G plus T plus YG infusates, respectively. Panel B represents the G infusate ( ) versus the T plus YG infusates ( π) ( P < 0.01). The standard errors of the means for progesterone were 0.25 and 0.19, and the standard errors of the means for estradiol were 0.2 and 0.2 pg/ml, for the curves representing the G infusate and the T plus YG infusates, respectively. and infusion of fat relative to the glucose infusion (2.42 vs ± 0.34 pg/ml; P = 0.023) decreased mean plasma estradiol concentrations during this 6-d period. Increased plasma concentrations of progesterone during the luteal phase for cows receiving the control infusion relative to energy infusions and for fat infusions relative to the glucose infusion (Figure 2) may be responsible for the subsequent pattern of plasma estradiol concentrations. Increased progesterone concentrations are likely to increase negative feedback of progesterone on estradiol production and subsequently decrease plasma concentrations of estradiol. The effects of yellow grease infusion during one period appeared to carry over into all subsequent periods when PGFM responses to an oxytocin

11 FAT INTAKE AND OVARIAN AND UTERINE FUNCTION 1325 Figure 3. Mean plasma 13,14-dihydro-15-keto-PGF 2a (PGFM) concentrations following an oxytocin challenge on d 15 of a synchronized estrous cycle for lactating Holstein cows receiving yellow grease ( o) or not receiving yellow grease ( ÿ) previously via abomasal infusion. Standard errors of the means for PGFM were 1.5 and 2.0 pg/ml for the curves for grease and nongrease, respectively. challenge were evaluated. Plasma PGFM data, therefore, were grouped into yellow grease treatment (period of yellow grease infusion plus all subsequent periods) and no yellow grease treatments (treatment periods prior to the yellow grease infusion). Regression of plasma concentrations of PGFM across time after oxytocin injection indicated that grease and no grease treatments differed ( P < 0.01) (Figure 3). Peak plasma PGFM concentrations were 47.9 and 80.0 pg/ml for the grease and no grease curves, respectively. Intraassay and interassay coefficients of variation for PGFM were 6.4 and 10.4%, respectively. An intravenous injection of oxytocin was expected to stimulate uterine production of PGF 2a and, subsequently, plasma concentration of PGFM. Infusion of yellow grease completely inhibited the postrise in plasma PGFM concentration that was detected after oxytocin injection in the no grease treatment periods. Comparable analyses for water, glucose, and tallow were not significant ( P > 0.05). Infusion of yellow grease delivered more linoleic acid to the small intestine than did tallow (60 vs. 8 g/ d). Linoleic acid is an essential fatty acid and a precursor for arachidonic acid synthesis and subsequent prostaglandin synthesis. Linoleic acid can be converted to dihomo-g-linolenic acid or arachidonic acid (21), either of which can act as substrate for prostaglandin endoperoxide synthase ( PES), the enzyme that catalyzes prostaglandin synthesis. Increased concentrations of substrate for PES can increase the rate of a suicide reaction (40) in which an inactive enzyme intermediate is formed or can result in peroxide accumulation and subsequent enzyme inactivity (46). Also, if dihomo-g-linolenic acid is the primary substrate metabolized by PES, one series prostaglandins would be produced, and production of two series prostaglandins would be reduced. Linoleic acid also acts as a competitive inhibitor of arachidonic acid metabolism by PES (43) and, subsequently, can decrease synthesis of the two series prostaglandins. Diets containing a high concentration of linoleic acid caused a decrease in arachidonic acid synthesis when fed to preruminant calves (22). Ovarian Dynamics Infusion of fat influenced the dynamics of the CL and dominant follicle. The maximum diameter of the CL tended to be 2 mm smaller, and disappearance of the CL was delayed, in cows infused with fat relative to cows infused with glucose (Table 7). As a result of delayed CL disappearance, the number of days until progesterone concentrations were 1 ng/ml tended to be prolonged for cows receiving fat infusions compared with those receiving the glucose infusion. This delay in CL disappearance and the delay in the reduction of progesterone to 1 ng/ml following injection of PGF 2a tended to be associated with the infusion of yellow grease rather than the infusion of tallow. Endogenous uterine secretion of PGF 2a from cows receiving the yellow grease infusion might have been decreased because of the inhibition of PES activity by linoleic acid, thus allowing the CL to remain functional slightly longer. Although exogenous PGF 2a (administered on d 15) initiates CL regression, a possible suppression of endogenous PGF 2a secretion by yellow grease would reduce the efficiency of achieving full CL regression. Furthermore, the observed reduction in estradiol that was due to fat (Figure 2) would further reduce the efficiency of CL regression because estradiol acts with PGF 2a to induce CL regression (43). Prostaglandins play a role in both the structural and functional regression of the CL. The first dominant follicle tended to emerge 2 d later when tallow was infused than when yellow grease was infused (Table 7). The delay in elevated ( 1 ng/ml) plasma progesterone concentrations following ovulation could have facilitated the earlier emergence of the first wave dominant follicle, its greater growth rate, and its larger maximum size for cows infused with yellow grease compared with those infused with tallow. Collectively, these alterations in follicular dynamics led to a larger first wave dominant follicle in cows infused with yellow grease. The first wave dominant follicle tended to lose its dominant influence 1 d earlier in control cows relative

12 1326 OLDICK ET AL. to cows receiving energy infusates (Table 7). The earlier loss of dominance for the first wave dominant follicle led to an earlier emergence of the second wave dominant follicle by 2 d in cows receiving the control infusate relative to those receiving energy infusates. Follicles were classified as class 1 (diameter, 5 mm), class 2 (diameter, 6 to 9 mm), or class 3 (diameter, 10 mm). The mean number of follicles within each class size during a synchronized estrous cycle was not affected by treatment [class 1, 9.3 ± 0.7; class 2, 1.3 ± 0.3; and class 3, 1.3 ± 0.1 (Figure 1B)]. Previous studies (26) indicated that follicle numbers and size can be influenced by the energy status of cows and fat supplementation. During the second follicular wave, different polynomial regression equations indicated a greater ( P < 0.01) recruitment or number of follicles that were 6 to 9 mm in diameter in cows infused with glucose relative to those infused with fat (Figure 4). This result suggests that fat decreased recruitment and reduced the pool from which to select the second dominant follicle. Maximum diameter and day of ovulation of the second dominant follicle were not affected by treatment. Because of the earlier emergence of the second wave dominant follicle in control cows, the control cows ovulated an older follicle. Understanding how these changes in follicular dynamics affect subsequent reproductive performance requires further investigation. CONCLUSIONS Increasing energy density of diets through supplemental fat may prove superior to increasing the proportion of dietary concentrate fed. Apparent digestibility of fatty acids was increased by fat infusion, suggesting that the supplemental fat was available to the cow. Abomasal infusions of fat tended to increase 4% FCM production and increased the efficiency of milk and FCM production relative to effects of carbohydrate (glucose) infusion. The type of fat supplemented may be important because efficiency of milk production was greater for cows infused with yellow grease than for cows infused with tallow; however, yellow grease decreased milk protein concentration relative to tallow. Further investigation of other supplemental fat sources is needed. Ovarian and uterine responses were dependent on both source of energy (carbohydrate vs. fat) and type of fat (tallow vs. yellow grease) that were infused TABLE 7. Characteristics (least squares means) of the corpus luteum (CL), first dominant follicle (DF1), first subordinate follicle (SF1), second dominant follicle (DF2), and second subordinate follicle (SF2) during a synchronized estrous cycle in lactating Holstein cows abomasally infused with water (W), glucose (G), tallow (T), and yellow grease (YG). 1First day the CL was visible as determined using ultrasonography. 2Plasma progesterone concentration. 3First day the CL was no longer visible as determined using ultrasonography. 4First day the diameter of DF1 exceeded the diameter of SF1 by at least 2 mm. 5First day follicles that were 6 to 9 mm in diameter increased in number. 6First day the diameter of DF2 exceeded the diameter of SF2 by at least 2 mm. Orthogonal contrast Infusate W vs. (G G vs. Item W G T YG SE +T+YG) (T + YG) T vs. YG P CL Emergence, 1 d Progesterone 1 ng/ml, 2 d CL Maximum diameter, mm CL Disappearance, 3 d Progesterone 1 ng/ml, 2 d DF1 Emergence, 4 d DF1 Mean growth rate, mm/d DF1 Stops growing, d DF1 Maximum diameter, mm DF1 Loses dominance, 5 d SF1 Maximum diameter, mm DF2 Emergence, 6 d DF2 Mean growth rate, mm/d DF2 Maximum diameter, mm DF2 Ovulated, d SF2 Maximum diameter, mm