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Nitrogen, fat, and lactose in milk were determined by an infrared method (National Milk Records PLC, Chippenham,. UK). Plasma concentrations of insulin were ...

Milk production, milk composition, and reproductive function of dairy cows fed different fats1 H. V. Petit1,5, R. J. Dewhurst2, J. G. Proulx1, M. Khalid3,6, W. Haresign3 and H. Twagiramungu4 1Dairy

and Swine Research and Development Centre, Agriculture and Agri-Food Canada, PO Box 90, Lennoxville, Quebec, Canada J1M 1Z3; 2Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Wales SY23 3EB, UK; 3Welsh Institute of Rural Studies, The University of Wales, Aberystwyth, Llanbadarn Campus, Aberystwyth, Ceredigion SY23 3AL, Wales, UK; and 4L’Alliance Boviteq Inc., SaintHyacinthe, Quebec, Canada J2S 7A9. Contribution number 695 from the Dairy and Swine Research and Development Centre. Received 10 October 2000, accepted 15 February 2001. Petit, H. V., Dewhurst, R. J., Proulx, J. G., Khalid, M., Haresign, W. and Twagiramungu, H. 2001. Milk production, milk composition, and reproductive function of dairy cows fed different fats. Can. J. Anim. Sci. 81: 263–271. Thirty-five non-gestating multiparous Holstein cows averaging 571 kg of BW (SE = 8) were allotted at 9 wk postpartum to one of two dietary fat supplements based on either Megalac® (Volac Ltd., Roston, Hertfordshire, UK) and solvent extracted flaxseed meal (MEGA) or whole flaxseed treated with formaldehyde (FLAX) to determine the effects on milk production and composition, follicular development, gestation rate, and fatty acid (FA) composition of blood. Cows were fed a total mixed diet based on ryegrass silage and fat supplements for ad libitum intake. The experiment was carried out between weeks 9 and 19 of lactation. Dry matter (DM) intake and change in body weight were similar for cows fed MEGA and FLAX. Milk production was higher for cows fed MEGA than for those fed FLAX (19.8 vs. 18.6 kg d–1) as was 4% fat-corrected milk yield (22.9 vs. 20.2 kg d–1). Increased fat mobilization could have contributed to increased milk yield when cows were fed MEGA compared with when they were fed FLAX as plasma concentrations of non-esterified FA and cholesterol increased more from weeks 9 to 19 of lactation for cows fed MEGA. Milk fat percentage tended (P = 0.06) to be greater for cows fed MEGA (4.62%) compared with those fed FLAX (4.37%). Milk protein percentage was higher for cows fed FLAX (3.09%) than for those fed MEGA (2.95%), indicating that formaldehyde protection of flaxseed was adequate to partly prevent ruminal degradability of protein in the seed. Milk fatty acid concentrations of C8:0, C10:0, C12:0, C14:0, C14:1, C18:0, C18:3, and C20:5 were higher for cows fed FLAX than for those fed MEGA while the inverse was observed for C16:0, C16:1, C18:1, and C18:2. Cows fed FLAX had lower blood concentrations of C16:0 than those fed MEGA. There was a significant interaction (P < 0.05) between week and diet for C18:0 and C18:2 with a decrease in C18:0 blood concentration for cows fed MEGA and an increase for those fed FLAX between weeks 9 and 19, while the inverse was observed for C18:2. Blood concentrations of C18:1 were similar for both treatments. Conception rate was significantly lower for cows fed MEGA (50.0%) compared to those fed FLAX (87.5%). Diet had no effect on the size of the largest and second largest follicles, or on the difference between the diameter of the largest and second largest follicles. The number of class 1 (1.09 vs. 0.86), 2 (1.33 vs. 0.86), and 3 (1.28 vs. 0.98) follicles was similar for MEGA and FLAX although the total number (3.70 vs. 2.70) of follicles tended (P = 0.09) to be greater for cows fed MEGA than for those fed FLAX. These data suggest that dietary FA have an effect on gestation rate, but this could not be explained by differences in follicle dynamics or number. However, additional trials with greater numbers of animals are needed to confirm the reproductive results. Key words: Dairy, flaxseed, milk production, reproduction, fatty acids Petit, H. V., Dewhurst, R. J., Proulx, J. G., Khalid, M., Haresign, W. et Twagiramungu, H. 2001. Milk production, milk composition, and reproductive function of dairy cows fed different fats. Can. J. Anim. Sci. 81: 263–271. Trente-cinq vaches multipares non-gestantes de race Holstein pesant une moyenne de 571 kg de poids vif (SE = 8) ont été réparties au hasard à 9 semaines après le vêlage entre deux rations contenant un supplément de gras à base de Megalac® (Volac Ltd., Roston, Hertfordshire, UK) et de tourteau de lin (MEGA) ou de graine de lin entière traitée à la formaldéhyde (FLAX) afin de déterminer les effets sur la production et la composition du lait, le développement folliculaire, le taux de gestation, et la composition en acides gras du sang. Les vaches ont reçu une ration totale mélangée d’ensilage de raygrass et de suppléments de gras pour une ingestion à volonté. L’expérience a été conduite entre les semaines 9 et 19 de lactation. L’ingestion de matière sèche et le changement de poids vif ont été semblables pour les vaches recevant le MEGA et le FLAX. La production de lait a été supérieure pour les vaches recevant le MEGA que pour celles recevant le FLAX (19.8 vs. 18.6 kg j–1) tout comme la production de lait corrigée à 4% de matière grasse (22.9 vs. 20.2 kg j–1). La plus grande mobilisation de tissu adipeux a pu contribuer à augmenter la production de lait lorsque les vaches ont reçu MEGA comparativement à celles ayant reçu FLAX tel que montré par les concentrations 5To

Abbreviations: ADF, acid detergent fibre; CL, corpus luteum; CP, crude protein; DM, dry matter; FA, fatty acids; FLAX, fat supplement based on whole flaxseed treated with formaldehyde; MEGA, fat supplement based on Megalac and solvent extracted flaxseed meal; NDF, neutral detergent fibre; PUFA, polyunsaturated fatty acids

whom correspondence should be addressed. This study was conducted while H. V. Petit was on leave at the Institute of Grassland and Environmental Research. 6Present address: Department of Farm Animal Medicine and Surgery, The Royal Veterinary College, Hawhshead Lane, North Mymms, Hatfield, HERTS, AL9 7TA, UK. 263

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CANADIAN JOURNAL OF ANIMAL SCIENCE plasmatiques d’acides gras non-estérifiés et de cholestérol qui ont augmenté davantage entre les semaines 9 et 19 de lactation pour les vaches recevant MEGA. Le pourcentage de gras a eu tendance (P = 0.06) a être supérieur pour les vaches recevant MEGA (4.62%) compativement à celles recevant FLAX (4.37%). Le pourcentage de protéine du lait a été supérieur pour les vaches recevant FLAX (3.09%) que pour celles recevant MEGA (2.95%), ce qui suggère que la protection à la formaldéhyde du lin a prévenu partiellement la dégradabilité ruminale de la protéine de la graine. Les concentrations en acides gras C8:0, C10:0, C12:0, C14:0, C14:1, C18:0, C18:3, et C20:5 ont été supérieures pour les vaches recevant FLAX que pour celles ayant reçu MEGA alors que l’inverse a été observé pour les concentrations de C16:0, C16:1, C18:1, et C18:2. Les vaches recevant FLAX ont eu une concentration sanguine en C16:0 plus faible que celles recevant MEGA. Il y a eu une interaction significative (P < 0.05) entre la semaine et la ration pour les C18:0 et C18:2 avec une diminution de la concentration sanguine en C18:0 pour les vaches recevant MEGA et une augmentation pour celles recevant FLAX entre les semaines 9 et 19, alors que l’inverse a été observé pour le C18:2. La concentration en C18:1 a été semblable pour les deux traitements. Le taux de conception a été significativement inférieur pour les vaches ayant reçu MEGA (50.0%) comparativement à celles ayant reçu FLAX (87.5%). Il n’y a eu aucune différence entre les rations pour ce qui est de la grandeur du plus grand follicule, deuxième plus grand follicule, et la dominance folliculaire (plus grand follicule-deuxième plus grand follicule). Le nombre de follicules de la classe 1 (1.09 vs. 0.86), 2 (1.33 vs. 0.86), et 3 (1.28 vs. 0.98) a été semblable pour MEGA et FLAX quoique le nombre total de follicules (3.70 vs. 2.70) a eu tendance à être (P = 0.09) plus grand pour les vaches recevant MEGA. Ces résultats suggèrent que les acides gras alimentaires ont un effet sur le taux de gestation mais ceci n’a pas pu être expliqué par les différences dans la dynamique folliculaire ou le nombre de follicules. Cependant, des essais additionnels avec un nombre plus important d’animaux sont nécessaires pour confirmer les effets de reproduction. Mots clés: Laitier, lin, production laitière, reproduction, acides gras

Feeding rumen-protected long-chain FA to high-producing cows can enhance energy density of the ration and energy intake without compromising rumen cellulolytic bacterial activity (Palmquist and Jenkins 1980). Moreover, milk FA composition may be modified by feeding polyunsaturated FA (PUFA) escaping ruminal digestion (Kennelly 1996). Dietary FA can also affect the reproductive health of the cow. For example, Sklan et al. (1991) reported that Ca soaps of FA increase pregnancy rate and reduce open days in highyielding cows, although Lucy et al. (1992) have shown that the type of FA and not additional energy provided by the FA stimulate the ovary and cause the development of large follicles. Membrane FA composition is modified by dietary FA (Thatcher et al. 1994), which might alter function of reproductive tissues. Linoleic acid is incorporated in the arachidonic acid pathway (Ashes et al. 1995) whereas α-linolenic acid leads to eicosapentaenoic acid formation (Béréziat 1978). Both arachidonic acid and eicosapentaenoic acid are precursors of prostaglandins but prostaglandins synthesized from eicosapentaenoic acid do not have the same biological activity as do those produced from arachidonic acid (Fly and Johnston 1990). Prostaglandins of the 2 series are derived from arachidonic acid while those of the 3 series can be formed from the eicosapentaenoic acid pathway (Abayasekara and Wathes 1999). Dietary FA from the omega-3 family reduce ovarian and endometrial synthesis of prostaglandin F2α, which may contribute to a reduction in embryonic mortality (Mattos et al. 2000). This suggests that linolenic acid feeding could decrease prostaglandin secretion of the 2 series or prostaglandin activity as suggested by Barnouin and Chassagne (1991), thus improving the fertility of cows. Ryegrass silage contains as much as 60% of linolenic acid as a percentage of total FA (Dewhurst and King 1998), which would encourage high forage systems to increase dietary linolenic acid content. The hypothesis of this experiment was that feeding a source rich in C18:3 would improve reproduction of dairy cows. The effects of feeding formaldehyde-treated flaxseed on milk production and com-

position, follicular development, gestation rate, and FA composition of blood in dairy cows fed high silage diets were also determined. MATERIALS AND METHODS Cows At 9 wk postpartum, 35 non-gestating multiparous Holstein cows from the Trawsgoed Farm (IGER, Aberystwyth, Wales, UK), averaging 571 kg of body weight (SE = 8 kg), were assigned to one of two dietary treatments. All cows calved within a 35-d period. Data on production (feed intake, milk production and composition, and FA composition of blood and milk) were collected from all cows between weeks 9 and 19 of lactation. Reproduction data (gestation rate and length of estrous cycle) were collected only from cows that had demonstrated normal estrous cycles as determined by repeated cyclicity in milk progesterone concentrations measured between weeks 9 and 19 postpartum. Five cows were considered as not having normal estrous cyles; there were two cows with anoestrus and one cow with a cyst in the MEGA treatment group and two cows with anoestrus in the FLAX treatment group. These five cows had persistently low milk progesterone concentrations and, thus, were not inseminated. Cows were housed in free stalls, fed individually using a Calan gate system (American Calan, Inc., Northwood, NH), and milked twice daily at 0600 and 1600 h. Milk production was recorded at every milking. Milk samples were obtained weekly from each cow for two consecutive milkings and analyzed separately to determine milk composition. Cows were weighed weekly. Body condition score was determined every week using a six-point scale (where 0 = emaciated and 5 = fat; Mulvany 1977). Animals were cared for according to the guidelines of the Canadian Council on Animal Care (1993). Feeding After parturition and before the beginning of the experiment at week 9 postpartum, all cows were fed a common diet that

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Table 1. Chemical composition of feed ingredientsz Item

Silage

Concentrate

Megalac®

Flaxseed meal

Flaxseed

pH DM (%) ADF (%) NDF (%) Gross energy (kcal g–1) CP (% of DM) Soluble N (% of N) NH3 N (% of N) Lactic acid (% of DM) Acetic acid (% of DM) Propionic acid (% of DM) Isobutyric acid (% of DM) Butyric acid (% of DM) Valeric acid (% of DM) Ether extract (% of DM) Fatty acid (% of total fatty acids) C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0

3.72 20.4 35.0 56.1 5.15 15.9 49.0 13.5 12.0 1.81 0.19 0 0.01 0 4.8

– 90.1 18.6 31.8 4.60 23.3 – – – – – – – – 7.3

– 96.9 – – 7.93 – – – – – – – – – 77.6

– 90.3 19.1 27.8 5.01 35.1 – – – – – – – – 10.4

– 90.7 29.7 47.7 6.15 26.3 – – – – – – – – 32.4

0 0 2.48 0.32 1.21 1.65 25.42 0.82 5.56 3.89 14.22 43.49 0.94

0.06 0.33 0.57 3.33 1.96 0 20.26 0.71 4.42 32.95 27.16 7.85 0.40

0.03 0.06 0.04 0.43 1.35 0 43.97 0.24 4.84 39.32 9.06 0.32 0.34

0 0 0.39 0.27 0.19 0 8.31 0.03 3.18 18.28 16.37 52.92 0.06

0 0 0.08 0 0.08 0 6.31 0.08 3.74 18.37 14.42 56.78 0.13

zMean

of seven 2-wk composite samples that were prepared from three weekly samples.

consisted of silage for ad libitum intake and 6 kg d–1 of a pelleted high-protein concentrate. Milk production and composition during weeks 7 and 8 of lactation were used as covariants for subsequent milk production and composition. Cows were introduced gradually to treatments over a 7-d period starting week 9 postpartum. All cows received a total mixed diet based on long-term perennial ryegrass silage and fat supplements offered for ad libitum intake (Table 1). The grass was harvested with a precision-chop forage harvester (Alois Pottinger MBH Mex VI PU457, Grieskirchen, Germany) and cut at a theoretical length of 5 cm from the primary growth of a stand of mostly ryegrass (> 90%, Lolium perenne), ensiled in bunkers, covered with plastic sheets, and weighted with straw bales. Silage was made between 3 and 6 June 1996. The chopped material was wilted for 24 h to approximately 21% DM and Add Safe (Trouw Nutrition Ltd., Northwich, UK), a mixture of 17% ammonium formate, 5% ammonium propionate, 38% formic acid and 11% propionic acid was applied at a rate of 5 L t–1 of fresh grass. The two dietary treatments (Table 2) consisted of fat supplements based on either solvent extracted flaxseed meal and Megalac® (Volac Ltd., Roston, Hertfordshire, UK) (MEGA), which is a Ca-soap of FA or whole flaxseed treated with formaldehyde (FLAX). There were 18 cows in the FLAX group and 17 cows in the MEGA group. Treatment of FLAX was carried out by adding 300 g of formalin per kg of whole flaxseeds to create pH reversible methylene bridges within the seed. Following treatment, the flaxseed was held for 5 d in curing vessels to

allow methyl linkages to form. Thus, the protected flaxseed was bagged and stored at ambient temperature after the curing phase. A pelleted concentrate (Table 1) was fed to all cows at a daily rate of 2.5 kg in one meal in the afternoon as top dress on the silage. Cows fed MEGA and FLAX diets received, respectively, 60 and 20 g d–1 of a mineral and vitamin mixture containing 14.5% P; 5.5% Mg; 9.75% Na; 12 mg kg–1 of Se; 100 mg kg–1 of Co; 450 mg kg–1 of I; 7000 mg kg–1 of Mn; 4500 mg kg–1 of Zn; 2750 mg kg–1 of Fe; 1000 mg kg–1 of Cu; 500 000 IU kg–1 of vitamin A; 100 000 IU kg–1 of vitamin D3; and 500 IU kg–1 of vitamin E. The two treatments were designed to provide similar crude protein (CP) and oil intakes and were formulated to meet requirements for cows averaging 550 kg of body weight, 25 kg of milk per day with 4.1% milk fat (Rumnut, The Ruminant Nutrition Program, A. T. Chamberlain, 1994 ed.). Diets were fed once daily with approximately 10% orts, and accumulated feed refusals were taken three times a week. Feed ingredients and total mixed diets were sampled three times a week, frozen, and composited on a 2-wk basis. Composited samples were mixed thoroughly and subsampled for chemical analyses. Measurements Blood was collected from all cows at weeks 0 and 10 of the experiment (weeks 9 and 19 postpartum, respectively) 3 h postfeeding. Blood was withdrawn from the jugular vein into vacutainer (Becton Dickinson Vacutainer Systems Europe BP 37, 38241 Meylan Cedex, France) tubes contain-

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Table 2. Ingredient and chemical composition of the two total mixed diets (DM basis except DM)z Composition

MEGA

FLAX

81.7 5.6 12.7 0

83.0 0 0 17.0

23.2 ± 0.94 16.7 ± 0.4 50.1 ± 1.4 30.9 ± 0.7 5.10 ± 0.04 9.3 ± 0.9 7.7 ± 0.7 3.5 ± 0.2

22.9 ± 1.4 17.0 ± 0.7 54.5 ± 0.7 31.8 ± 1.1 5.13 ± 0.03 10.0 ± 1.1 7.1 ± 0.5 3.9 ± 0.4

Fatty acid (% of total fatty acids) C10:0 0.84 ± 0.11 C12:0 0.30 ± 0.02 C14:0 1.37 ± 0.07 C14:1 0 C16:0 32.13 ± 0.72 C16:1 0.30 ± 0.05 C18:0 3.66 ± 0.08 C18:1 25.27 ± 0.82 C18:2 12.49 ± 0.05 C18:3 23.21 ± 0.98 C20:0 0.43 ± 0.02

0.78 ± 0.16 0.09 ± 0.02 0.39 ± 0.11 0.40 ± 0.10 11.17 ± 1.22 0.22 ± 0.07 4.65 ± 0.36 14.58 ± 0.64 14.67 ± 0.80 52.74 ± 3.34 0.31 ± 0.05

Ingredient (% of DM) Silage Megalac® Flaxseed meal Formaldehyde whole flaxseed Nutrienty DM (%) CP (%) NDF (%) ADF (%) Energy (kcal g–1) Ether extract (%) Ash (%) Starch (%)

zMEGA = fat supplement based on Megalac® and flaxseed meal and FLAX

= fat supplement based on formaldehyde treated whole flaxseed. yMean of seven 2-wk composite samples that were prepared from three weekly samples ± standard deviation (SD).

ing heparin for determination of insulin and glucose, into vacutainer (Becton Dickinson) tubes containing EDTA for non-esterified FA and FA analysis, and into vacutainer tubes (Becton Dickinson) without preservative for cholesterol analysis. The plasma and serum were separated and frozen at –20°C for subsequent analysis. Milk was collected three times a week from the start of the experiment until 45 d after artificial insemination for progesterone analysis. Timing of ovulations and corpora lutea formation were estimated from sustained elevations in milk progesterone concentration (at least 7 d above 3 µg L–1) as described by Lucy et al. (1992). Ovulation was defined as occurring 5 d prior to first elevation in milk progesterone or was determined exactly for cows observed in estrus (ovulation = estrus day + 1). Estrous cycle length was estimated as the interval between successive ovulations. Cows were observed for signs of estrus after milking for a 30-min period two times daily from the beginning of the experiment and started to be inseminated after 6 wk on the diets (wk 15 of lactation). Cows were inseminated 12 h after the end of standing heat. The same two technicians performed the inseminations and semen came from a single ejaculate of two bulls, ensuring that equal numbers of cows from each treatment group were bred to each bull. Ultrasound scanning was performed on day 45 after AI to confirm pregnancy. Conception rate was defined as the proportion of cows that were detected in estrus and inseminated that were pregnant on day 45 post AI.

Ovaries of eight cows per dietary treatment were examined by ultrasonography using a Concept\MCV ultrasound scanner equipped with a linear array 7.5 MHz probe (Dynamic Imaging Ltd, Livingston, Scotland, UK) in midmorning on every second day during the second estrous cycle of the experiment as determined by milk progesterone. Examinations involved removal of faecal matter from the rectum, insertion of the probe into the rectum, and positioning of the probe adjacent to each ovary. Size and number of ovarian follicles > 3 mm were recorded on detailed follicular maps designed to identify specific large follicles (> 5 mm) on repeated days. Procedures were similar generally to those described by De La Sota et al. (1993). In this way, the size of the largest and second largest follicles could be followed during the preovulatory period. Cystic follicles were always associated with low milk progesterone concentrations, thus were not considered as the largest follicles. Follicles were grouped into three diameter classes for analyses: class 1 (3.0 to 4.9 mm), class 2 (5.0 to 9.9 mm), and class 3 (≥ 10 mm). Size, number, and position of corpus luteum (CL) and large luteinized follicles also were recorded. Chemical Analysis Dry matter of silage was determined according to Dewar and McDonald (1981). Dry matter of total mixed diets and other feed ingredients was obtained by drying at 100°C for 48 h. An aqueous extract (20 g of silage in 100 mL of water) was used to determine concentrations of D- and L-lactate (kit No. 139 084, Boehringer Mannheim Ltd., Lewes, East Sussex, UK), ammonia (kit No. 66-50, Sigma-Aldrich Co., Ltd., Poole, Dorset, UK) with a discrete analyzer (FP-901 M Chemistry Analyzer, Labsystems Oy, Helsinki, Finland) and pH using a pH meter (FisherBrand Hydrus 400, Orion Research Inc, Beverly, MA). Silage volatile FA were determined by gas chromatography (ATI Unicam, Cambridge, UK) on an aliquot of the extract using 2-ethyl-butyric acid as the internal standard chromatography (Fussell and McCalley 1987). Soluble N was calculated as the portion dissolved after a 2-h incubation in artificial saliva at 40°C (Merry et al. 1987). Ether extraction was obtained using Soxtec equipment (Perstorp Analytical Ltd., Maidenhead, Berkshire, UK) with the acid hydrolysis ether extract method incorporating the additional acid hydrolysis and extraction step described in Thomas et al. (1988). Crude protein (N × 6.25) determinations were done by the Kjeldahl method. Neutral detergent fibre (NDF) was determined using the modified method with amylase described by Van Soest et al. (1991) while acid detergent fibre (ADF) was analyzed according to Van Soest and Wine (1967). Gross energy of freeze-dried silages was measured with an adiabatic bomb calorimeter (Sanyo Gallenkamp, Leicester, UK). Concentrations of plasma glucose (kit No. 6, Sigma-Aldrich Ltd., Poole, Dorset, UK), plasma non-esterified FA (kit 9075401; Wako Pure Chemical Industries, Osaka, Japan), and serum cholesterol (kit No. 352, Sigma-Aldrich Ltd., Poole, Dorset, UK) were analyzed by colorimetric methods. Nitrogen, fat, and lactose in milk were determined by an infrared method (National Milk Records PLC, Chippenham, UK). Plasma concentrations of insulin were determined by

PETIT ET AL. — FLAXSEED AND REPRODUCTION

radioimmunoassay using a kit (Life Screen Ltd., Watford, UK). All samples were measured in one assay and the intraassay coefficient of variation was < 14% and the inter-assay variation was < 8%. Concentrations of progesterone in whole milk (Ridgeway Science Ltd, Alvington, UK) were measured by enzyme immunoassay (Groves et al. 1990) in a single assay every week for samples collected in the same week, and intra- and inter-assay coefficients of variation were 4.2 and 7.5%, respectively. A total of 13 assays was carried out. The limit of detection with the progesterone assay was 0.5 ng mL–1. Fatty acid composition of blood, milk, and feed ingredients was carried out using the one-step saponification-methylation method of Sukhija and Palmquist (1988). Samples were introduced to the gas chromatograph via a split injector onto a cross-linked polyethylene glycol column (Innowax 30 m × 0.32 mm × 0.5 µm). A temperature gradient from 60 to 230°C effected the separation of FA methyl esters, which were detected by a flame ionization detector. Fatty acids were identified according to their retention time (AI-450 Software; Dionex Ltd, Camberley, Surrey, UK) using a reference standard (ME61 Quantitative; Greyhound Chromatography, West Birkenhead, Merseyside, UK). Cis-10-nonadecenoic acid (C19:1) was added to samples as an injection standard just prior to loading onto the gas chromatograph. Statistical Analysis All results were subjected to least squares ANOVA for a randomized complete block design with two treatments using the general linear models procedure of the SAS Institute, Inc. (1985). Data on milk production and milk composition were analyzed using the mean values for the preceeding 2 wk as covariate. Data on blood composition were analyzed as repeated measurements across time. Significance was declared at P < 0.05 unless otherwise stated. Data on follicular development were analyzed from days 14 to 21 of the estrous cycle. This part of the estrous cycle includes three successive events representing: first, the end of the luteal phase or diestrous with CL development and important secretion of progesterone; second, the follicular phase or proestrous with PGF2α secretion, CL regression, decrease progesterone concentration, and selection of a preovulatory follicle; and third, the estrus and ovulation with the lowest progesterone concentration. Synthesis of PGF2α is responsible for luteolysis, thus leading to embryo loss and return into heat. Our hypothesis was that feeding a source of linolenic acid would inhibit PGF2α synthesis and improve the maintenance of gestation. Student’s t tests were used to determine the effect of treatments on length of estrus cycle, size of CL, the difference in size between the largest and the second largest follicle, and class and number of follicles. The number and percentage of cows pregnant were tested using a chi-square test. RESULTS AND DISCUSSION Ether extract supplied by the concentrate portion represented 5.4 and 6% of the total mixed diet for MEGA and FLAX, respectively (data not shown). Percentages of CP, NDF, and ADF were very similar in both diets (Table 2). Dry matter

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Table 3. Dry matter intake (DMI), body weight (BW) change and covariant-adjusted milk production and milk composition of Holstein cows fed between weeks 9 and 19 of lactation a total mixed diet containing Megalac® and flaxseed meal (MEGA) or formaldehyde-treated whole flaxseed (FLAX)z Item DMI, kg d–1 % of BW BW change (g d–1) Milk production (kg

MEGA

FLAX

13.4 2.37

13.6 2.41

–61 d–1)y

4% fat-corrected milk yield (kg d–1)y

–4

SE 0.2 0.03 54

19.8a

18.6b

0.4

22.9a

20.2b

0.4

Milk composition (%) Fatx Protein Lactose

4.62 2.95b 4.78

4.37 3.09a 4.77

0.09 0.04 0.03

Milk components production (kg d–1) Fat Protein Lactose

0.92a 0.58 0.95

0.81b 0.57 0.89

0.02 0.01 0.03

Somatic cell count, x103 ml–1

300

181

54

zLeast squares means with pooled standard error (SE). yCovariant-adjusted fat-corrected milk production at weeks

7 and 8 of lactation. xP = 0.06. a, b Means within a row with a different letter differ (P < 0.05).

intake, expressed in kilogram per day or as a percentage of body weight, and change in body weight were similar for cows fed MEGA and FLAX (Table 3). Untreated whole flaxseed is readily accepted by dairy cows and feeding up to 15% of the total DM as flaxseed has no effect on DM intake (Kennelly and Khorasani 1992). There was no difference in tail and loin body condition score, and in change of body condition score of tail and loin (data not shown). Similar results were reported for cows fed extruded soybeans and rolled sunflower seeds (Schingoethe et al. 1996) or 4.5% Ca salts and nonenzymatically browned soybeans (Abel-Caines et al. 1998). Change in body weight was not affected by treatment (Table 3) although all cows lost weight. A loss in body weight was not expected based on a study that used a diet containing a similar silage to concentrate ratio as was used in our study (Aston et al. 1998). However, the loss in body weight can be explained by the lower silage quality in the present experiment; ryegrass was harvested in June in our study compared with the third week of May in the study by Aston et al. (1998). Milk production (Table 3) was lower than that reported by Aston et al. (1998) for cows fed similar silage to concentrate ratio, which may be a result of the more-mature silage fed in the present experiment. Milk production and 4% fatcorrected milk yield were higher for cows fed MEGA than for those fed FLAX. Schingoethe et al. (1996) reported no difference in yields of milk and 4% fat-corrected milk

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between cows fed either extruded soybeans or rolled sunflower seeds. Kennelly and Khorasani (1992) found no difference in milk yield and milk fat when cows were fed no flaxseed or up to 15% of the total DM as flaxseed although flaxseed decreased milk protein. In another experiment, however, the same authors (Khorasani and Kennelly 1994) observed a decrease in DM intake and milk yield when whole flaxseed was fed at 10% of the total DM. In this last experiment, flaxseed feeding produced relatively small changes in the concentration of C18:3 in milk, indicating that extensive biohydrogenation of PUFA occurred in the rumen, thus probably depressing fibre digestion and feed intake. Protein concentration in milk was higher for cows fed FLAX than for those fed MEGA (Table 3), which disagrees with the decrease observed by Kennelly and Khorasani (1992). Protein concentration is usually lower for cows supplemented with Ca long-chain FA such as Megalac® (Shaver 1990) although Schingoethe et al. (1996) found a trend (P = 0.06) for higher protein concentration when cows were fed sunflower seeds compared with when they were fed extruded soybeans. A depression in milk protein concentration is frequently associated with dietary lipid supplementation; however, daily protein production may be unchanged as supplemental fat tends to have a positive effect on milk yield (Kennelly 1996). The formaldehyde treatment applied to flaxseed could have prevented ruminal protein degradability and increased the amount of protein secreted in milk. Milk lactose percentage was not affected by the diet. Daily yield of fat was higher for cows fed MEGA than for those fed FLAX, which reflects the greater milk production and fat percentage (P = 0.06) for cows fed MEGA. There was no difference between diets in yield of protein and lactose. Milk somatic cell count was similar for both diets, as previously reported for cows fed either extruded soybeans or rolled sunflowers as dietary fat sources (Shaver 1990). Milk fatty acid concentrations (Table 4) of C8:0, C10:0, C12:0, C14:0, C14:1, C18:0, C18:3, and C20:5 were higher for cows fed FLAX than for those fed MEGA while the inverse was observed for C16:0, C16:1, C18:1, and C18:2. Protection of canola seed with aldehyde also decreased the proportion of C16:0 and increased that of C18 FA (Ashes et al. 1992). In general, modification of milk FA concentrations were similar to those reported for cows fed up to 15% of flaxseed in the total DM (Kennelly and Khorasani 1992). Grundy and Denke (1990) reported that C16:0 and C18:0 mono- and PUFA elevate and lower, respectively, serum cholesterol in humans. Cows fed FLAX would, therefore, produce milk to better meet human requirements for food leading to decreased blood cholesterol concentrations. There was no difference between treatments for C18 total, but the C16:0 to C18 total ratio was greater for cows fed MEGA. Concentrations of short-chain FA were higher for cows fed FLAX and medium-chain FA concentrations were lower than for cows fed MEGA. There was no difference in long-chain FA concentrations between cows fed MEGA and FLAX. Individual short- and medium-chain FA (C14:0, C16:0, and C16:1) concentrations in blood decreased from

Table 4. Milk fatty acid composition of Holstein cows fed between weeks 9 and 19 of lactation a total mixed diet containing Megalac® and flaxseed meal (MEGA) or formaldehyde-treated whole flaxseed (FLAX)z Item

MEGA

C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:5 C18 total C16:0/C18 total SCFAy MCFA LCFA

2.29b 1.70b 2.03b 2.44b 8.97b 0.72b 35.63a 1.72a 13.69b 27.86a 1.77a 0.89b 0.20b 44.21 0.86a 8.47b 45.32a 44.42

FLAX (% of total fatty acids)

SE

2.55a 2.02a 2.66a 3.16a 11.02a 0.86a 31.68b 1.54b 15.37a 26.07b 1.56b 1.22a 0.22a 44.21 0.77b 10.39a 43.57b 44.44

0.04 0.04 0.08 0.09 0.21 0.02 0.50 0.04 0.30 0.36 0.04 0.04 0.01 0.50 0.02 0.23 0.45 0.50

zLeast square means with pooled standard error (SE). ySCFA = short-chain fatty acids (4:0 to 12:0), MCFA = medium-chain fatty

acids (14:0 to 16:0), and LCFA = long-chain fatty acids (18:0 to 20:5) a, b Means within a row with a different letters differ (P < 0.05).

week 9 to week 19 of lactation, while there was a general increase in long-chain FA concentrations (Table 5). Fatty acid composition of milk and blood would mainly reflect influences of the diet. For instance, the increase from week 9 to week 19 of lactation would result from an increase in dietary fat content as cows fed supplemental dietary fat have a greater proportion of long-chain FA than unsupplemented cows (Shaver 1990). Cows fed FLAX had lower C16:0 concentration than those fed MEGA. There was a significant interaction (P < 0.05) between week and diet for C18:0 and C18:2 with a decrease in C18:0 blood concentration for cows fed MEGA and an increase for those fed FLAX between weeks 9 and 19, while the inverse was observed for C18:2. Concentration of C18:1 was similar for both treatments. Concentrations of C18:3 and C18 total were higher during week 19 than during week 9 of lactation, and they were higher for cows fed FLAX than for those fed MEGA. Feeding flax increases the proportion of linolenic acid in milk fat (Kennelly 1996). The C16:0 to C18 total ratio was higher during week 9 than week 19, and it was higher for cows fed MEGA than for those fed FLAX. Concentration of plasma non-esterified FA increased from week 9 to week 19 for cows fed MEGA, whereas it remained similar for those fed FLAX, resulting in a significant interaction between week and diet (Table 5). Greater non-esterified FA concentration is related to increased body fat mobilization (Roberts et al. 1981), suggesting that increased fat mobilization could have contributed to increase milk yield when cows were fed MEGA compared with when they were fed FLAX (Table 3). There was a trend (P = 0.12) for an interaction between week and fat for blood

PETIT ET AL. — FLAXSEED AND REPRODUCTION

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Table 5. Blood composition of Holstein cows fed between weeks 9 and 19 of lactation a total mixed diet containing Megalac® and flaxseed meal (MEGA) or formaldehyde-treated whole flaxseed (FLAX)z MEGA Item Non-esterified fatty acids (µEq L–1)y Insulin (µU mL–1) Glucose (mM)x Fatty acid (% of total fatty acids) C14:0x C16:0wx C16:1x C18:0wxy C18:1 C18:2wy C18:3wx C18 totalwx C16:0/C18 totalwx Cholesterol (mg 100 mL–1) totalwxy HDLxy LDLwx zLeast squares means with yFat × week interaction (P xWeek effect (P < 0.05). wFat effect (P < 0.05).

FLAX

wk 9

wk 19

wk9

wk 19

SE

109b 13.7 3.50

151a 11.2 3.96

146.9a 9.3 3.58

133ab 11.5 3.84

7.1 1.2 0.04

2.48 18.02 1.83 19.53ab 15.87 32.46b 9.81 77.66 0.2562 254c 193c 61

1.87 17.10 1.60 15.85c 15.92 35.70a 11.96 79.42 0.2357 418a 296a 122

2.14 17.04 1.99 18.60b 16.21 33.09b 10.93 78.83 0.2419 259c 204c 55

2.11 15.09 1.46 20.00a 14.47 27.26c 19.61 81.34 0.2038 327b 249b 78

0.08 0.23 0.05 0.29 0.32 0.46 0.54 0.25 0.0038 10 7 6

pooled standard error (SE). < 0.05).

a–c Means within a row with a different letters differ (P < 0.05).

glucose concentration as a result of greater increase in glucose concentration between week 9 and week 19 for cows fed MEGA compared with those fed FLAX. Greater nonesterified FA and lower glucose concentrations in blood are associated with greater negative energy balance (Bauman and Currie 1980). Milk production was greater for cows fed MEGA than for those fed FLAX although DM intake was similar, suggesting greater negative energy balance for the former. As greater negative energy balance is associated with reduced reproductive performance (Spicer et al. 1990), this could partly explain the lower conception rate for cows fed MEGA compared with those fed FLAX in the present experiment. Lower glucose concentration is also related to lower ruminal protein degradability (Garcia-Bojalil et al. 1998), which would further support the efficacy of formaldehyde to protect flaxseed protein against the attack of rumen microbes. According to Spicer et al. (1993), insulin is a powerful stimulator of follicular cell development. However, in the present experiment, it is unlikely that insulin was the only factor involved in follicle development as cows fed MEGA and FLAX had similar plasma insulin concentration. Plasma concentrations of total and HDL cholesterol increased from week 9 to week 19 of lactation, but the increase was greater for cows fed MEGA than for those fed FLAX, which resulted in a significant interaction between week and diet. Fat supplementation is known to increase blood cholesterol (Spicer et al. 1993; Garcia-Bojalil et al. 1998), which would explain the increase observed in the present experiment from week 9 to week 19 as cows began to receive fat from week 9. Moreover, the greater increase in blood cholesterol for cows fed MEGA compared with those fed FLAX cor-

roborate the depressing effect of omega-3 FA as supplied by flax on blood cholesterol already reported in humans (Cunnane 1995). Concentration of LDL cholesterol was greater in week 19 than week 9 of lactation and it was greater for cows fed MEGA than for those fed FLAX. It has been suggested that improved conception rate could be a result of increased concentrations in plasma cholesterol (Spicer et al. 1993), although this hypothesis was not supported by the results of the present experiment. In fact, cows fed FLAX had lower plasma cholesterol concentration and a better conception rate than those fed MEGA. Other studies have reported no relationship between cholesterol concentrations in blood and reproductive measures (Ferguson et al. 1990; Spicer et al. 1990). Length of estrous cycle was similar for the two diets (Table 6). Conception rates were significantly higher for cows fed FLAX (87.5%) than for those fed MEGA (50.0%). There was no effect of diet on the diameter of the largest and second largest follicules, or on the difference in diameter between the largest and second largest follicle. Furthermore, there was no effect of diet on the number of class 1, 2, and 3 follicles. However, the total number of follicles tended (P = 0.09) to be greater for cows fed MEGA than for those fed FLAX. The CL was similar for cows fed MEGA and those fed FLAX. The area under the curve for milk progesterone concentration tended (P = 0.14) to be greater for cows fed FLAX than for those fed MEGA. Better conception rate for cows fed FLAX compared with those fed MEGA could result from different prostaglandin synthesis. In fact, linolenic acid in flaxseed uses the eicosapentaenoic acid metabolic pathway while FA in MEGA uses the arachidonic acid pathway (Cunnane 1995) and it is

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Table 6. Reproduction data of Holstein cows fed between weeks 9 and 19 of lactation a total mixed diet containing Megalac® and flaxseed meal (MEGA) or formaldehyde-treated whole flaxseed (FLAX)z Item Length of estrous cycle (d) Conception at first AI no/no % Follicle diameter (mm)y Largest (F1) Second largest (F2) F1-F2 Size classes of follicles (number)y 3.0 to 4.9 mm 5.0 to 9.9 mm ≥ 10.0 mm Total number of folliclesy Corpus luteum (CL) diameter, mm Milk progesterone (µg L–1) Mean value Peak value Area under the curve

MEGA

FLAX

P

SE

21.7

22.8

0.36

1.1

7/14 50.0b

14/16 87.5a

0.04

13.9 8.0 5.8

13.4 6.8 5.6

0.78 0.29 0.90

1.1 0.7 1.0

1.09 1.33 1.28 3.70 21.6

0.86 0.86 0.98 2.70 20.3

0.57 0.32 0.21 0.09 0.43

0.30 0.38 0.16 0.41 1.0

11.1 26.8 249.9

12.4 35.2 302.4

0.23 0.17 0.14

0.8 4.6 26.7

zLeast squares means with pooled standard error (SE). yMean between d 14 and 21 of the estrous cycle.

a, b Means within a row with a different letters differ (P < 0.05).

known that eicosapentaenoic acid inhibits prostaglandin synthesis (Spicer et al. 1993). Therefore, ingestion of linolenic acid contained in flaxseed could potentially inhibit PGF2α synthesis (Cunnane 1995). Moreover, flaxseed was treated with formaldehyde in the present experiment, which would have prevented biohydrogenation of some linolenic acid by rumen microbes as shown by the increased in linolenic acid content in milk (Table 4), potentially increasing eicosapentaenoic acid formation and furthermore enhancing the depressing effect of eicosapentaenoic acid on prostaglandins synthesis. Thatcher et al. (1997) has shown that PGF2α secretion is decreased in dairy cows fed fish meal. In fact, fish meal, which would lead to eicosapentaenoic acid and docosahexaenoic acid formation, has been shown to increase gestation rate of dairy cows and to alter CL regression as shown by greater plasma concentrations of progesterone (Burke et al. 1997). This would agree with the tendency observed in the present experiment for greater milk progesterone concentration, expressed as the area under the curve, for cows fed FLAX compared with those fed MEGA. However, it is not known if the greater conception rate observed for cows fed FLAX in the present experiment is a result of a decrease in embryo mortality or better fertilization of the ova as pregnancy was confirmed only once at day 45 post-AI. More research is required to determine the reasons for better conception rate for cows fed a source rich in omega-3 FA. The potential to improve reproduction of dairy cows through dietary manipulation is an exciting concept and needs to be further addressed. CONCLUSIONS Feeding Megalac® increased milk yield and milk fat percentage compared with feeding formaldehyde-treated flaxseed, although milk protein was higher with formaldehyde-treated flaxseed. Both sources of FA resulted in simi-

lar feed intake, but the increase in concentration of plasma NEFA was greater for cows fed calcium salts compared with those fed formaldehyde-treated flaxseed, suggesting that increased fat mobilization could have contributed to increased milk yield when cows were fed calcium salts. Conception rate was lower when Megalac® was fed, which would corroborate the fact that cows were in greater negative energy balance when fed calcium salts compared with when they were fed formaldehyde-treated flaxseed, as negative energy balance is associated with reduced reproductive performance. These data suggest that fat utilization could differ according to the type of dietary FA. ACKNOWLEDGEMENTS The authors thank J. Tweed, P. King, and W. J. Fisher for technical assistance and the dairy barn staff at the Trawsgoed farm in Wales for helping maintain the cows. Abayasekara, D. R. E. and Wathes, D. C. 1999. Effects of altering dietary fatty acid composition on prostaglandin synthesis and fertility. Prostaglandins Leukot. Essent. Fatty Acids 61: 275–287. Abel-Caines, S. F., Grant, R. J., Klopfenstein, T. J., Winowiski, T. and Barney, N. 1998. Influence of nonenzymatically browned soybeans on ruminal fermentation and lactational performance of dairy cows. J. Dairy Sci. 81: 1036–1045. Ashes, J. R., Welch, P. S., Gulati, S. K., Scott, T. W., Brown, G. H. and Blakely, S. 1992. Manipulation of the fatty acid composition of milk by feeding protected canola seeds. J. Dairy Sci. 75: 1090–1096. Ashes, J. R., Fleck, E. and Scott, T. W. 1995. Dietary manipulation of membrane lipids and its implications for their role in the production of second messengers. Pages 373–386 in W. V. Engelhardt, S. Leonhard-Marek, B. Breves, and D. Giesecke, eds. Ruminant physiology: Digestion, metabolism, growth and reproduction. Proc. 8th Int. Symp. Ruminant Physiology, Ferdinand Enke Verlag, Germany.

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