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Effect of organic or inorganic trace mineral supplementation on follicular response, ovulation, and embryo production in superovulated Angus heifers
Animal Reproduction Science 106 (2008) 221–231
Effect of organic or inorganic trace mineral supplementation on follicular response, ovulation, and embryo production in superovulated Angus heifers G. Cliff Lamb a,∗ , Daniel R. Brown a , Jamie E. Larson a , Carl R. Dahlen b , Nicolas DiLorenzo d , John D. Arthington c , Alfredo DiCostanzo d a
North Central Research and Outreach Center, University of Minnesota, 1861 Highway 169E, Grand Rapids, MN 55744, USA b Northwest Research and Outreach Center, University of Minnesota, 2900 University Avenue, Crookston, MN 56716, USA c Range Cattle Research and Education Center, University of Florida, 3401 Experiment Station, Ona, FL 33865, USA d Department of Animal Science, Haecker Hall, 1364 Eckles Avenue, St. Paul, MN 55108, USA Received 19 July 2006; accepted 12 April 2007 Available online 20 April 2007
Abstract We determined whether source of trace mineral supplementation prior to embryo collection affected embryo production and quality. Angus half-sibling heifers (n = 20) originating from a common herd were assigned to three treatment groups using a 3 × 3 latin square design replicated in time (3×) and space (6× complete and 1× incomplete): (1) heifers received no added mineral to their diet (control; n = 53); (2) heifers received a commercially available organic mineral supplement (organic; n = 52); or (3) heifers received an all inorganic mineral supplement (inorganic; n = 55). All heifers had ad libitum access to hay and were fed a supplement containing corn and soybean meal. Treatments were initiated 23 days prior to embryo recovery. Heifers were given a 45-day adaptation period of no mineral supplementation before initiating a new treatment. Ovarian structures were evaluated using transrectal ultrasonography to determine the presence and number of follicles and CL on each ovary. The mean number of recovered ova/embryos was similar among treatments (4.1 ± 0.7, 3.8 ± 0.7, and 3.3 ± 0.7 for control, inorganic, and organic treatments, respectively), the number of unfertilized oocytes was greater (P < 0.05) for inorganic (2.3 ± 0.5) and control (1.6 ± 0.5) treated heifers than organic (0.4 ± 0.4) treated heifers. No differences among treatments existed for the number of degenerate or transferable embryos, but individual heifer influenced the total number of ∗
Corresponding author. Tel.: +1 218 327 4490; fax: +1 218 327 4126. E-mail address: [email protected] (G.C. Lamb).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.04.007
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embryos/ova, unfertilized ova, and transferable embryos recovered. We conclude that heifer accounted for the greatest differences in embryo production and quality. Source of trace mineral supplementation did not significantly alter embryo number or quality in superovulated purebred Angus heifers fed a well-balanced diet, meeting all trace mineral requirements. © 2007 Elsevier B.V. All rights reserved. Keywords: Cattle-embryo transfer; Beef heifers; Mineral supplementation
1. Introduction Appropriate supplementation of trace minerals is essential for maintaining growth, immune function, and reproductive performance in beef cattle (Graham, 1991; Underwood and Suttle, 1999; Baily et al., 2001). The form of mineral supplementation also may affect individual animal performance. Organic trace minerals differ from inorganic forms as a result of their chemical association with an organic ligand. The resulting mineral-organic ligand combination can result in the production of one of several classes of organic trace minerals, which are available in the animal feeding industry and include chelates, proteinates, and complexes (AAFCO, 2000). Previous analysis of organic minerals has indicated that these may be of greater bioavailability compared to their inorganic counterparts (Brown and Zeringue, 1994). Published data comparing organic with inorganic trace mineral supplementation in an embryo transfer program do not exist, yet current dogma among embryologists indicates that donor cows fed supplements with organic minerals yield greater quantities of embryos that are of enhanced quality than donors supplemented with inorganic minerals or no minerals during superovulation. The objective of the present study was, therefore, to determine whether the source of supplemental whole trace inorganic and organic mineral sources, prior to embryo collection, altered embryo production and quality. 2. Materials and methods This study was performed between January 2000 and April 2003. Angus half-sibling, heifers (n = 20) originating from a common herd (Mundhenke beef, Lewis, KS) that were born between 8 August and 24 October 1999, were acclimated to the North Central Research and Outreach Center, Grand Rapids, MN from 21 August to 1 December 2000. Heifers were then assigned randomly to one of three treatment groups using a 3 × 3 latin square design in time (3×) and space (6× complete and 1× incomplete): (1) heifers received no added mineral to their diet (control; n = 53); (2) heifers received 108 g daily of an organic mineral (organic; Albion Cattle Breeder Pak, Des Moines, IA, USA; n = 52); or (3) heifers received 108 g daily of an all inorganic mineral (inorganic; inorganic Breeder Pak, Des Moines, IA, USA; n = 55). Latin squares were replicated fully six times (n = 3) and incompletely once (n = 2) in space, and fully replicated three times in time (Table 1). A 25-mg injection of PGF (Lutalyse, Pfizer Animal Health, New York, NY) was administered on Day 23 (i.e. 23 days prior to embryo collection) at which point daily, individual feeding of an isonitrogenous, isocaloric supplement containing corn, soybean meal, and mineral, was initiated and fed until the day of embryo collection (Tables 2 and 3). Both organic and inorganic mineral sources were blended into the supplement and offered at a rate of 108 g/animal/day. In addition, within a pen, heifers were provided ad libitum access to hay during the supplementation period (Table 2). After harvesting embryos, heifers were provided no mineral
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Table 1 Experimental design indicating the assignment of three treatment groups using a 3 × 3 latin square design. in time (3×) and space (6× complete and 1× incomplete)a
a Heifers (A–T) were assigned to three treatment groups (C = control; I = inorganic; O = organic) using a 3 × 3 latin square design. in time (3×) and space (6× complete and 1× incomplete).
supplement for a minimum of 45 days and then reassigned to receive a different treatment. The animals utilized in this experiment were cared for by acceptable practices and all procedures were approved by the University of Minnesota Institute for Animal Use and Care Committee. All heifers were monitored for signs of estrus using Kamar® Heatmount® detectors (Kamar, Inc., Steamboat Springs, CO), but regardless of estrus status, on Day 16 all heifers received a 1 mg injection of estradiol cypionate (ECP, Pfizer Animal Health, New York, NY) and a Controlled Internal Drug Releasing (CIDR) insert containing 1.9 g of progesterone (Pfizer Animal Health, New York, NY). From Day 12 to 9, heifers received 29 mg of follicle stimulating hormone (FSHp, batch 9109, Sioux Biochemical, Sioux Center, IA) administered twice daily in the following doses 6.25, 5.25, 4.50, 3.75, 3.00, 2.50, 2.00, 1.75 mg. On Day 9 heifers received two 25 mg injections of PGF given 12 h apart and the CIDR was removed at the second PGF injection. Regardless of estrous response, all heifers were inseminated artificially at 36, 48, and 60 h after CIDR removal (Fig. 1). Semen used for artificial insemination was collected from two bulls in four ejaculates and was frozen according to Certified Semen Services Inc. standards (a subsidiary of National Association of Animal Breeders, Columbia, MO). Semen from a single bull from each ejaculate was assigned randomly to each embryo collection. On Day 0, embryos were recovered by a single embryo technician using a nonsurgical embryo collection procedure and were evaluated under a stereomicroscope. All embryos were assigned a developmental stage and quality grade according to standards set forth by the International Embryo Transfer Society (Savoy, IL). Developmental stage codes were: 3 = early morula; 4 = morula; 5 = early blastocyst; and, 6 = blastocyst. Quality codes were: 1 = symmetrical and spherical embryo mass with individual blastomeres that were uniform in size, color, and density with at least 85% of the cellular material intact (excellent or good); 2 = moderate irregularities in overall shape of embryonic mass or in size, color, and density of individual cells with at least 50% of the cellular material intact (fair); 3 = major irregularities in shape of the embryonic mass or size, color, and density of individual cells with at least 25% of the cellular material intact (poor); 4 = dead or degenerating; and 5 = unfertilized.
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Table 2 Average daily intake of supplement and hay and mineral composition of total diets consumed for heifers assigned to inorganic, organic, and control treatments (DM basis) Treatmentsa
Item
Control Supplementb
Inorganic
Organic
0.55 2.80
0.55 2.81
(kg)
SBM Corn Hayc Total intake Mineral compositiond (%) Calcium Phosphorus Magnesium Potassium Sodium Sulfur PPM Cobalt Copper Iron Manganese Molybdenum Zinc
0.46 2.86 10.74 ± 0.17 14.06 ± 0.17
10.60 ± 0.17 13.95 ± 0.17
10.83 ± 0.17 14.19 ± 0.17
0.90 0.36 0.24 1.86 0.03 0.21
0.89 0.35 0.38 1.92 0.03 0.22
0.89 0.36 0.37 1.90 0.04 0.23
0.60 12.94 196.66 54.65 3.11 51.36
0.70 54.77 216.63 141.66 3.12 187.45
0.99 50.60 219.27 156.02 3.18 221.18
a Heifers received either 108 g of organic mineral (Albion Breeder Pak, Des Moines, IA), 108 g inorganic mineral (Inorganic Breeder Pak, Des Moines, IA, USA using the following sources: CaCO3 , CaHPO4 , MgO, MnO, MnS, CuSO4 , CuO, CoCO3 , ZnS, ZnO4 , KCl, NaCl), or no mineral daily for the 23 days prior to embryo collection. b Heifers received a daily supplement fed individually to each heifer containing mineral, soybean meal (SBM) and corn. c Heifers were allowed ad libitum access to hay. Intakes were determined by monitoring group intake for each treatment during a 7-day collection period. d Average daily individual mineral consumption based on total diet consumed by individual heifers.
On Days 12, 7, and 0, ovaries were scanned via transrectal ultrasonography (5-MHz intrarectal transducer, Aloka 500 V, Corometrics, Wallingford, CT) to determine the presence, number, and size of all follicles greater than 5 mm in diameter. The diameter of each follicle was calculated as the mean of the vertical and horizontal diameters. In addition, the presence and number of corpora lutea (CL) on each ovary were determined (Fig. 1). All ultrasonographic images were recorded on ovarian maps at the time of ultrasound for later review and analysis. Procedure MIXED was used to analyze noncategorical data (SAS Inst. Inc., Cary, NC). The model was used to analyze the total number of embryos collected, embryo stage and quality, degenerate embryos, unfertilized embryos, transferable embryos, follicles present on Day 7, CL present on Day 0, follicles from which ovulation had not occurred by Day 0, and the number of follicles determined on Day 7 for various size categories included treatment, period, square (replication in time), repetition (replication in space), heifer, and all two-way interactions with treatment. Heifer within treatment was used as the error term for period and square, whereas the residual error was used for repetitions and all interactions. All tests were determined to be significant when the P value for main effects was <0.05.
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Table 3 Ovarian follicle stimulation in heifers receiving inorganic, organic or no mineral after superovulation with follicle stimulating hormone Treatmentsa
Item
heifersb
All treated No. of heifers
P-value
Control
Inorganic
Organic
53
55
52
11.78 ± 1.11 7.97 ± 1.13 2.15 ± 0.53 0.66 ± 0.19
13.48 ± 1.09 8.21 ± 1.11 1.13 ± 0.52 0.59 ± 0.19
11.83 ± 1.09 6.97 ± 1.11 1.81 ± 0.52 0.55 ± 0.19
0.494 0.730 0.419 0.928
6.44 ± 0.89 1.75 ± 0.39
6.59 ± 0.87 1.99 ± 0.38
4.93 ± 0.87 1.74 ± 0.38
0.376 0.880
(n ± S.E.)
folliclesc
No. of >7 mm >9 mm >11 mm >13 mm
No. of corpora lutead No. of unovulated folliclese Responding heifersf No. of heifers No. of follicles >7 mm >9 mm >11 mm >13 mm No. of corpora lutead No. of unovulated folliclese
27
35
31
13.78 ± 1.46 9.76 ± 1.40 1.64 ± 0.76 0.50 ± 0.16
13.99 ± 1.20 8.75 ± 1.16 1.76 ± 0.62 0.77 ± 0.13
13.17 ± 1.35 7.09 ± 1.31 2.11 ± 0.71 0.56 ± 0.15
0.885 0.351 0.882 0.379
10.07 ± 1.13 2.45 ± 0.59
8.88 ± 0.94 2.69 ± 0.48
8.52 ± 1.06 2.85 ± 0.55
0.588 0.882
a Heifers received either 108 g of organic mineral, 108 g inorganic mineral, or no mineral for the 23 days prior to embryo collection. b All heifers receiving FSH (excluding 20 heifers from period five). c Determined 7 days before embryo collection by transrectal ultrasonography. d Determined on the day of embryo collection by transrectal ultrasonography. e Determined on the day of embryo collection by transrectal ultrasonography. f Only heifers responding to FSH (i.e., containing at least one CL at the time of embryo collection).
Overall, 180 embryo recoveries were scheduled (60 per treatment); however, 29 potential recoveries were removed from analysis associated with embryo numbers and quality because the recovery catheter was unable to be passed through the cervix or insufficient media was retrieved to warrant a successful recovery. In addition, ovarian ultrasound data during period five were not
Fig. 1. Timeline demonstrating superstimulation protocol for heifers assigned to inorganic, organic, or no mineral treatments. ECP, estradiol cypionate; FSH, follicle stimulating hormone; PGF, PGF2α ; CIDR, controlled internal drug release containing 1.9 g progesterone.
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collected because of damage to the ultrasonography machine. After initial analysis of all treated heifers, those that failed to respond to superstimulation of ovarian follicular development (defined as heifers that did not have at least one CL present at the time of embryo recovery on Day 0) were removed and a second analysis was performed using the same model. Pearson correlations were calculated among variables using the CORR procedure of SAS to determine correlations between: number of embryos recovered, degenerate embryos, unfertilized embryos, transferable embryos, and number and size class of follicles present on Day 7, CL present on Day 0, and unovulated follicles on Day 0. 3. Results Daily dry matter intake of hay was similar among treatments during the supplementation phase (Table 2). Among all heifers, daily dry matter intake was 14.1 ± 1.8 kg (mean ± S.D.). Of all the heifers assigned to a treatment, 63 69, and 56% of the control, inorganic, and organic treatment groups, respectively, had a CL on Day 12, when FSH was initiated. In addition, the response to superovulation was similar among mineral treatments (51, 64, and 60% for control, inorganic, and organic treatments, respectively). Data for response of heifers to superovulation treatments is summarized in Table 3. The number of follicles >7, >9, >11, and >13 mm on Day 7 was similar among all treatments. In addition, on Day 0 the number of CL and the number of follicles from which ovulation had not occurred was similar among all treatments. When heifers that failed to respond to superovulation treatments (i.e., there was no evidence of a CL on Day 0) were removed for separate analysis, the mean number of follicles on Day 7 and the number of CL on Day 0 increased, but no treatment differences were noted. Embryo number and quality data are summarized in Table 4. Among all heifers, the mean number of recovered embryos was similar among treatments (4.1 ± 0.7, 3.8 ± 0.7, and 3.3 ± 0.7 for control, inorganic-, and organic-treated heifers, respectively). The number of unfertilized oocytes was greater (P < 0.05) for inorganic (2.3 ± 0.4) and control (1.6 ± 0.4) groups than organictreated (0.4 ± 0.4) group. The number of degenerate and transferable embryos was similar among treatments. When data from heifers that failed to respond to superovulation were eliminated, the mean number of embryos increased to 6.5 ± 1.2 for control, 5.0 ± 1.0 for inorganic-treated, and 5.0 ± 1.1 for organic-treated heifers, respectively. Organic (0.5 ± 0.5) and inorganic (0.3 ± 0.4) treatment groups tended (P = 0.087) to have fewer degenerate embryos than control treated heifers (1.7 ± 0.5), whereas the number of unfertilized ova tended (P = 0.084) to be greater for inorganic (3.1 ± 0.7) than organic treatment (1.1 ± 0.7) groups. The number of unfertilized ova in the control (2.0 ± 0.8) group remained intermediate. As anticipated, there were differences (P < 0.001) among heifers in the total number of embryos recovered, which ranged from 0.3 ± 1.6 to 11.6 ± 1.9 embryos per recovery. Similarly, there were differences (P < 0.001) among heifers in the number of transferable embryos, unfertilized oocytes, and quality grade one and two embryos. In addition, there was a trend for a difference (P = 0.063) and a difference (P < 0.05) existed among heifers in the number of degenerate and quality grade three embryos, respectively. The total number of follicles >7 mm on Day 7 also differed (P < 0.001) among heifers, ranging from 5.4 ± 2.3 to 20.2 ± 2.3 follicles per recovery. At embryo recovery (Day 0), the average number of CL per heifer also differed (P < 0.001) and ranged from 0.7 ± 1.9 to 16.0 ± 1.9 CL per heifer. In addition, the number of follicles from which ovulation did not occur and were present on Day 0 also differed (P < 0.01) among heifers, ranging from 0.6 ± 0.8 to 4.4 ± 0.8 per heifer.
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Table 4 Embryo/ova production in heifers receiving inorganic, organic or no mineral after superovulation with follicle stimulating hormone Treatmentsa
Item
P-value
Control
Inorganic
Organic
(n ± S.E.) All treated No. of heifers No. of embryos/ova recovered No. of degenerate/cleaved No. of unfertilized No. of transferable Grade 1 Grade 2 Grade 3
49 4.09 ± 0.74 0.94 ± 0.33 1.56 ± 0.46c 1.61 ± 0.53 1.07 ± 0.44 0.52 ± 0.18 0.00 ± 0.04
51 3.79 ± 0.74 0.28 ± 0.33 2.34 ± 0.46c 1.14 ± 0.53 0.81 ± 0.44 0.31 ± 0.18 0.04 ± 0.04
51 3.29 ± 0.73 0.19 ± 0.31 0.44 ± 0.44c 2.13 ± 0.50 1.51 ± 0.42 0.58 ± 0.17 0.03 ± 0.04
0.462 0.266 0.024 0.445 0.555 0.569 0.550
Responding heifersd No. of heifers No. of mean embryos/ova recovered No. of degenerate/cleaved No. of unfertilized No. of transferable
23 6.54 ± 1.14 1.73 ± 0.49 1.97 ± 0.75 2.82 ± 0.79
31 4.99 ± 1.00 0.33 ± 0.43 3.10 ± 0.65 1.58 ± 0.69
30 4.99 ± 1.05 0.45 ± 0.46 1.12 ± 0.68 3.38 ± 0.73
0.549 0.087 0.084 0.150
heifersb
a
Heifers received either 108 g of organic mineral, 108 g inorganic mineral, or no mineral for the 23 days prior to embryo collection. b All heifers receiving FSH (excluding 29 heifers eliminated because of poor recovery of media). c Uncommon means within a row differ (P < 0.05) d Only heifers responding to FSH and when embryo retrieval was successful (i.e., containing at least one CL at the time of embryo collection).
Although there were no period × treatment interactions for any embryo characteristic variables, there were period effects during the study. The mean number of embryos collected per recovery per period did not differ among periods and ranged from 2.0 ± 0.9 to 5.4 ± 1.0 embryos per collection. Differences (P < 0.05) existed among periods in the number of unfertilized oocytes, transferable embryos, and quality grade two embryos. A tendency (P = 0.063) was noted for quality grade one embryos to differ among periods. The mean number of follicles >7 mm on Day 7 differed (P < 0.01) among periods and ranged from 7.8 ± 1.4 to 15.7 ± 1.4 follicles. The average number of CL on Day 0 was 5.8 ± 6.6 (mean ± S.D.) per period and was similar among periods; however, the average number of follicles from which ovulation did not occur ranged from 0.8 ± 0.5 to 3.8 ± 0.5 and was different (P < 0.05) among periods. Correlation coefficients between factors affected by superstimulation (number of follicles, size of follicles, and number of CL) and embryo collection factors (embryo number and quality) are summarized in Table 5. The most significant (P < 0.0001) correlation (r = 0.773) occurred between the number of CL at the day of embryo recovery and total number of collected embryos. However, the number of CL was also correlated with the number of degenerate, unfertilized oocytes, and transferable embryos (0.414; 0.428, and 0.422, respectively; P < 0.001). The number of follicles >7 mm on Day 7 had a greater correlation (r = 0.648; P < 0.0001) with total embryos recovered than the number of follicles >9, >11, or >13 mm on Day 7. Interestingly, number of follicles from which ovulation did not occur were correlated (r = 0.412; P < 0.0001) with the number of unfertilized ova.
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Table 5 Pearson correlation coefficients between the number of embryos for each embryo classification and ovarian follicle sizes, number of corpora lutea and follicles from which ovulation did not occur Embryo classificationb
Degenerate/cleaved Unfertilized Transferable Total ova/embryos a b c d e f g
Follicle sizesa >7 mm
>9 mm
>11 mm
>13 mm
0.288e
0.311f
0.480g 0.270e 0.648g
0.206 0.214e 0.426g
0.166 −0.088 0.194 0.148
−0.068 −0.046 −0.112 −0.139
Corpora luteac
Unovulated folliclesd
0.414f 0.428g 0.422g 0.773g
−0.071 0.412g −0.107 0.239
Determined seven days before embryo collection by transrectal ultrasonography. Classified according to the International Embryo Transfer Society (Savoy, IL). Determined on the day of embryo collection by transrectal ultrasonography. Determined on the day of embryo collection by transrectal ultrasonography. Correlation exists (P < 0.01). Correlation exists (P < 0.001). Correlation exists (P < 0.0001).
4. Discussion Current dogma in the embryo transfer community is that feeding organic mineral rather than inorganic mineral enhances the number of embryos and/or the quality of embryos recovered. Analysis of organic minerals has indicated that they may be of greater bioavailability compared with inorganic counterparts (Brown and Zeringue, 1994). Therefore, the potential exists for the enhanced bioavailability of organic trace minerals to enhance reproductive efficiency and potentially increase embryo number and quality. The organic product utilized in this study is a chelated (i.e., when a mineral is bound to amino acids with at least two bonds from each amino acid) mineral designed for donor cows in an embryo transfer program, with the recommendation that donors receive 108 g of the mineral daily for a minimum of 21 days prior to embryo recovery. The present study was designed to test the hypothesis that supplementing donor heifers with an organic mineral for three weeks prior to embryo recovery would enhance the number and quality of embryos collected in a superovulation program compared to those receiving an inorganic mineral or no mineral supplementation during the same period. As expected, not all heifers responded to superstimulation, but there was no difference among treatments in the percentage of heifers that did respond. It is well established that ovulation rate can be influenced by environmental factors such as nutrition and has become apparent that these nuritional effects are mediated by a direct action at the level of the ovary, involving insulin, insulin-like growth factors (IGF) I and II, and their binding proteins among other factors (Hunter et al., 2004). These factors can also affect the quality of the oocyte and consequently, embryo development and survival (Hunter et al., 2004). Bioavailability of organic minerals is greater than inorganic minerals and could potentially alter the nutrition-related factors affecting the number of oocytes available for ovulation or the quality of oocytes. However, in the current study mineral treatments started only 23 days prior to embryo collection. This may not have been sufficient time for the ovaries to be impacted by effects, if any, of mineral source to alter ovulation rates. Numerous studies have demonstrated nutritional effects on follicular and oocyte development and viability. Donors that had less than ideal energy intake prior to and during superovulation, had more follicles and improved embryo quality (Armstrong et al., 2001). Further, when oocytes were collected from hyperinsulinemic heifers oocyte quality was compromised (Adamiak et al., 2005)
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and sheep fed energy-restricted diets had increased embryo quality compared to high-energy diets (Nolan et al., 1998). Heifers fed a greater-energy diet were observed to have increased peripheral IGF-I and enhanced the intrafollicular IGF-I, resulting in increased growth rates of dominant follicles; however, cows receiving greater-protein diets had decreased oocyte quality, due in part to increased plasma urea concentrations (Armstrong et al., 2001). In the present study all heifers were maintained on a moderate energy and protein diet, which met but did not exceed NRC (1996) recommendations. The primary difference among treatments was trace mineral source. Perhaps these donors having been fed a well-balanced diet meeting recommendations for all nutrients negated the potential positive effects of feeding a more bioavailable mineral such as the organic mineral fed in the present study. We are unaware of data demonstrating local effects of trace mineral source on oocyte maturation or quality and follicular development. However, reports have indicated that the use of organic mineral has variable effects on reproductive performance in beef females. When beef cows were fed an organic mineral an increase in artificial insemination pregnancy rates during the subsequent breeding season were noted (Stanton et al., 2000). Further, young cows were noted to have increased reproductive performance after receiving organic mineral, whereas no effect was noted in mature cows (Arthington and Swenson, 2004). In sows, embryo and fetal survival were enhanced and incidence of return to estrus was reduced when sows received an organic mineral. In contrast, feeding supplemental organic or inorganic Cu did not enhance reproductive performance (Muehlenbein et al., 2001) and feeding excess organic or inorganic mineral reduced reproductive performance (Olson et al., 1999). These inconsistent data are similar with results from the present study. In the present study there was no difference in the mean number of embryos/ova recovered among the control, inorganic, or organic treatment groups, but there were subtle differences or tendencies for differences in the number of degenerate, unfertilized, and transferable embryos collected. Heifers receiving organic mineral had a tendency for a greater number of transferable embryos than inorganic treated heifers, but the increase appeared to be a result of an increase in the number of quality grade two embryos, rather than an increase in grade one embryos. However, no difference was detected between organic and control treatment groups. When eliminating the data for non-responding heifers, no differences in transferable embryos were detected. We hypothesized that feeding organic mineral would enhance embryo numbers. To increase embryo numbers, ovarian follicular dynamics and CL numbers would need to be altered to reflect the change in embryo numbers. Therefore, ultrasonographic examinations were performed to determine whether mineral treatment affected follicle development and ovulation rates. However, no differences were detected in follicle numbers, size of follicles, the percentage of follicles from which ovulation occurred, or the number of CL that developed demonstrating that organic or inorganic mineral supplementation during the 23 days prior to embryo recovery, did not appear to influence growth and ovulation from follicles. Even though all the heifers were half-siblings, specific heifers had greater embryo yields, more follicular growth, and ovulated from more follicles than other heifers. These data demonstrated that regardless of treatment, individual heifer had a greater impact on embryo quality and number than mineral treatment. In addition, period appeared to have a greater effect on embryo production than mineral treatment, primarily related to climatic differences among periods, which are known to have impacts on reproductive efficiency in cattle (de la Sota et al., 1998; Rensis and Scaramuzzi, 2003). The greatest correlation was demonstrated between number of embryos and the number of CL present on Day 0. Embryologists utilize palpation of CL at the time of recovery to predict total number of embryos and the correlation determined in this study (r = 0.773) was similar to
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that previously reported (Donaldson, 1985). However, an interesting observation of the current study was the number of unovulated follicles at the time of embryo recovery, which correlated (r = 0.412) to unfertilized oocytes. To our knowledge this is the first observation demonstrating this correlation and warrants additional research. Clearly a number of mechanisms are involved in mediating the effects of diet on ovarian function and the formulation of diets designed to optimize fertility in cattle must consider the effects of nutrient supply on follicular growth and oocyte quality. In the present study, heifers received a balanced diet to meet NRC (1996) recommendations and perhaps masked potential conclusive positive or negative effects of organic compared with inorganic mineral supplementation. However, heifer and period accounted for the greatest differences in embryo production and quality. Following 23 days of mineral supplementation, which was the feeding protocol used in this experiment, neither organic nor inorganic minerals appeared to significantly alter embryo numbers and quality. Acknowledgements Appreciation is expressed to John Mundhenke, Mundhenke Beef, Lewis, KS for use of heifers to conduct this study. The authors thank Albion Advanced Nutrition, Clearfield, UT for partial financial support and donation of mineral products and Pfizer Animal Health, New York, NY, for donation of PGF2α (Lutalyse® ) and CIDR inserts. Appreciation also is expressed to S. Logan, A. Perez-Salazar, A. Spell, K. Thielen, and R. Wasson for their assistance with data collection. References AAFCO. 2000. Official Publication. Association of American Feed Control Officials, Inc., Oxford, IN. Adamiak, S.J., Mackie, K., Watt, R.G., Webb, R., Sinclair, K.D., 2005. Impact of nutrition on oocyte quality: cumulative effects of body composition and diet leading to hyperinsulinemia in cattle. Biol. Reprod. 73, 918–926. Armstrong, D.G., McEvoy, T.G., Baxter, G., Robinson, J.J., Hogg, C.O., Woad, K.J., Webb, R., Sinclair, K.D., 2001. Effect of dietary energy and protein on bovine follicular dynamics and embryo production in vitro: associations with the ovarian insulin-like growth factor system. Biol. Reprod. 64, 1624–1632. Arthington, J.D., Swenson, C.K., 2004. Effects of trace mineral source and feeding method on the productivity of grazing Braford cows. Prof. Anim. Sci. 20, 155–161. Baily, J.D., Ansotegui, R.P., Paterson, J.A., Swenson, C.K., Johnson, A.B., 2001. Effects of supplementing combinations of inorganic and complexed copper on performance and liver mineral status of beef heifers consuming antagonists. J. Anim. Sci. 79, 2926–2934. Brown, T.F., Zeringue, L.K., 1994. Laboratory evaluations of solubility and structural integrity of complexed and chelated trace mineral supplements. J. Dairy Sci. 77, 181–189. de la Sota, R.L., Burke, J.M., Risko, C.A., Moreira, F., DeLorenzo, M.A., Thatcher, W.W., 1998. Evaluation of timed insemination during summer heat stress in lactating dairy cattle. Theriogenology 49, 761–770. Donaldson, L.E., 1985. Estimation of superovulation response in donor cows. Vet. Rec. 117, 33–34. Graham, T.W., 1991. Element deficiencies in cattle. Vet. Clin. North Am. Food Anim. Pract. 7, 153–215. Hunter, M.G., Robinson, R.S., Mann, G.E., Webb, R., 2004. Endocrine and paracrine control of follicular development and ovulation rate in farm species. Anim. Reprod. Sci. 82–83, 461–477. Muehlenbein, E.L., Brink, D.R., Deutcher, G.H., Carlson, M.P., Johnson, A.B., 2001. Effects of inorganic and organic copper supplemented to first-calf cows on cow reproduction and calf health and performance. J. Anim. Sci. 79, 1650–1659. Nolan, R., O’Callaghan, D., Duby, R.T., Lonergan, P., Boland, M.P., 1998. The influence of short-term nutrient changes on follicle growth and embryo production following superovulation in beef heifers. Theriogenology 50, 1263–1274. Olson, P.A., Brink, D.R., Hickok, D.T., Carlson, M.P., Schneider, N.R., Deutscher, G.H., Adams, D.C., Colburn, D.J., Johnson, A.B., 1999. Effects of supplementation of organic and inorganic combinations of copper, cobalt, manganese, and zinc above nutrient requirement levels on postpartum two-year-old cows. J. Anim. Sci. 77, 522–532.
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Rensis, F.D., Scaramuzzi, R.J., 2003. Heat stress and seasonal effects on reproduction in dairy cows: a review. Theriogenology 60, 1139–1151. Stanton, T.L., Whittier, J.C., Geary, T.W., Kimberling, C.V., Johnson, A.B., 2000. Effects of trace mineral supplementation on cow-calf performance, reproduction, and immune function. Prof. Anim. Sci. 16, 121–127. Underwood, E.J., Suttle, N.F., 1999. The mineral nutrition of livestock, 3rd. CABI Publishing Co., New York.
Effect of organic or inorganic trace mineral supplementation on follicular response, ovulation, and embryo production in superovulated Angus heifers G. Cliff Lamb a,∗ , Daniel R. Brown a , Jamie E. Larson a , Carl R. Dahlen b , Nicolas DiLorenzo d , John D. Arthington c , Alfredo DiCostanzo d a
North Central Research and Outreach Center, University of Minnesota, 1861 Highway 169E, Grand Rapids, MN 55744, USA b Northwest Research and Outreach Center, University of Minnesota, 2900 University Avenue, Crookston, MN 56716, USA c Range Cattle Research and Education Center, University of Florida, 3401 Experiment Station, Ona, FL 33865, USA d Department of Animal Science, Haecker Hall, 1364 Eckles Avenue, St. Paul, MN 55108, USA Received 19 July 2006; accepted 12 April 2007 Available online 20 April 2007
Abstract We determined whether source of trace mineral supplementation prior to embryo collection affected embryo production and quality. Angus half-sibling heifers (n = 20) originating from a common herd were assigned to three treatment groups using a 3 × 3 latin square design replicated in time (3×) and space (6× complete and 1× incomplete): (1) heifers received no added mineral to their diet (control; n = 53); (2) heifers received a commercially available organic mineral supplement (organic; n = 52); or (3) heifers received an all inorganic mineral supplement (inorganic; n = 55). All heifers had ad libitum access to hay and were fed a supplement containing corn and soybean meal. Treatments were initiated 23 days prior to embryo recovery. Heifers were given a 45-day adaptation period of no mineral supplementation before initiating a new treatment. Ovarian structures were evaluated using transrectal ultrasonography to determine the presence and number of follicles and CL on each ovary. The mean number of recovered ova/embryos was similar among treatments (4.1 ± 0.7, 3.8 ± 0.7, and 3.3 ± 0.7 for control, inorganic, and organic treatments, respectively), the number of unfertilized oocytes was greater (P < 0.05) for inorganic (2.3 ± 0.5) and control (1.6 ± 0.5) treated heifers than organic (0.4 ± 0.4) treated heifers. No differences among treatments existed for the number of degenerate or transferable embryos, but individual heifer influenced the total number of ∗
Corresponding author. Tel.: +1 218 327 4490; fax: +1 218 327 4126. E-mail address: [email protected] (G.C. Lamb).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.04.007
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embryos/ova, unfertilized ova, and transferable embryos recovered. We conclude that heifer accounted for the greatest differences in embryo production and quality. Source of trace mineral supplementation did not significantly alter embryo number or quality in superovulated purebred Angus heifers fed a well-balanced diet, meeting all trace mineral requirements. © 2007 Elsevier B.V. All rights reserved. Keywords: Cattle-embryo transfer; Beef heifers; Mineral supplementation
1. Introduction Appropriate supplementation of trace minerals is essential for maintaining growth, immune function, and reproductive performance in beef cattle (Graham, 1991; Underwood and Suttle, 1999; Baily et al., 2001). The form of mineral supplementation also may affect individual animal performance. Organic trace minerals differ from inorganic forms as a result of their chemical association with an organic ligand. The resulting mineral-organic ligand combination can result in the production of one of several classes of organic trace minerals, which are available in the animal feeding industry and include chelates, proteinates, and complexes (AAFCO, 2000). Previous analysis of organic minerals has indicated that these may be of greater bioavailability compared to their inorganic counterparts (Brown and Zeringue, 1994). Published data comparing organic with inorganic trace mineral supplementation in an embryo transfer program do not exist, yet current dogma among embryologists indicates that donor cows fed supplements with organic minerals yield greater quantities of embryos that are of enhanced quality than donors supplemented with inorganic minerals or no minerals during superovulation. The objective of the present study was, therefore, to determine whether the source of supplemental whole trace inorganic and organic mineral sources, prior to embryo collection, altered embryo production and quality. 2. Materials and methods This study was performed between January 2000 and April 2003. Angus half-sibling, heifers (n = 20) originating from a common herd (Mundhenke beef, Lewis, KS) that were born between 8 August and 24 October 1999, were acclimated to the North Central Research and Outreach Center, Grand Rapids, MN from 21 August to 1 December 2000. Heifers were then assigned randomly to one of three treatment groups using a 3 × 3 latin square design in time (3×) and space (6× complete and 1× incomplete): (1) heifers received no added mineral to their diet (control; n = 53); (2) heifers received 108 g daily of an organic mineral (organic; Albion Cattle Breeder Pak, Des Moines, IA, USA; n = 52); or (3) heifers received 108 g daily of an all inorganic mineral (inorganic; inorganic Breeder Pak, Des Moines, IA, USA; n = 55). Latin squares were replicated fully six times (n = 3) and incompletely once (n = 2) in space, and fully replicated three times in time (Table 1). A 25-mg injection of PGF (Lutalyse, Pfizer Animal Health, New York, NY) was administered on Day 23 (i.e. 23 days prior to embryo collection) at which point daily, individual feeding of an isonitrogenous, isocaloric supplement containing corn, soybean meal, and mineral, was initiated and fed until the day of embryo collection (Tables 2 and 3). Both organic and inorganic mineral sources were blended into the supplement and offered at a rate of 108 g/animal/day. In addition, within a pen, heifers were provided ad libitum access to hay during the supplementation period (Table 2). After harvesting embryos, heifers were provided no mineral
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Table 1 Experimental design indicating the assignment of three treatment groups using a 3 × 3 latin square design. in time (3×) and space (6× complete and 1× incomplete)a
a Heifers (A–T) were assigned to three treatment groups (C = control; I = inorganic; O = organic) using a 3 × 3 latin square design. in time (3×) and space (6× complete and 1× incomplete).
supplement for a minimum of 45 days and then reassigned to receive a different treatment. The animals utilized in this experiment were cared for by acceptable practices and all procedures were approved by the University of Minnesota Institute for Animal Use and Care Committee. All heifers were monitored for signs of estrus using Kamar® Heatmount® detectors (Kamar, Inc., Steamboat Springs, CO), but regardless of estrus status, on Day 16 all heifers received a 1 mg injection of estradiol cypionate (ECP, Pfizer Animal Health, New York, NY) and a Controlled Internal Drug Releasing (CIDR) insert containing 1.9 g of progesterone (Pfizer Animal Health, New York, NY). From Day 12 to 9, heifers received 29 mg of follicle stimulating hormone (FSHp, batch 9109, Sioux Biochemical, Sioux Center, IA) administered twice daily in the following doses 6.25, 5.25, 4.50, 3.75, 3.00, 2.50, 2.00, 1.75 mg. On Day 9 heifers received two 25 mg injections of PGF given 12 h apart and the CIDR was removed at the second PGF injection. Regardless of estrous response, all heifers were inseminated artificially at 36, 48, and 60 h after CIDR removal (Fig. 1). Semen used for artificial insemination was collected from two bulls in four ejaculates and was frozen according to Certified Semen Services Inc. standards (a subsidiary of National Association of Animal Breeders, Columbia, MO). Semen from a single bull from each ejaculate was assigned randomly to each embryo collection. On Day 0, embryos were recovered by a single embryo technician using a nonsurgical embryo collection procedure and were evaluated under a stereomicroscope. All embryos were assigned a developmental stage and quality grade according to standards set forth by the International Embryo Transfer Society (Savoy, IL). Developmental stage codes were: 3 = early morula; 4 = morula; 5 = early blastocyst; and, 6 = blastocyst. Quality codes were: 1 = symmetrical and spherical embryo mass with individual blastomeres that were uniform in size, color, and density with at least 85% of the cellular material intact (excellent or good); 2 = moderate irregularities in overall shape of embryonic mass or in size, color, and density of individual cells with at least 50% of the cellular material intact (fair); 3 = major irregularities in shape of the embryonic mass or size, color, and density of individual cells with at least 25% of the cellular material intact (poor); 4 = dead or degenerating; and 5 = unfertilized.
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Table 2 Average daily intake of supplement and hay and mineral composition of total diets consumed for heifers assigned to inorganic, organic, and control treatments (DM basis) Treatmentsa
Item
Control Supplementb
Inorganic
Organic
0.55 2.80
0.55 2.81
(kg)
SBM Corn Hayc Total intake Mineral compositiond (%) Calcium Phosphorus Magnesium Potassium Sodium Sulfur PPM Cobalt Copper Iron Manganese Molybdenum Zinc
0.46 2.86 10.74 ± 0.17 14.06 ± 0.17
10.60 ± 0.17 13.95 ± 0.17
10.83 ± 0.17 14.19 ± 0.17
0.90 0.36 0.24 1.86 0.03 0.21
0.89 0.35 0.38 1.92 0.03 0.22
0.89 0.36 0.37 1.90 0.04 0.23
0.60 12.94 196.66 54.65 3.11 51.36
0.70 54.77 216.63 141.66 3.12 187.45
0.99 50.60 219.27 156.02 3.18 221.18
a Heifers received either 108 g of organic mineral (Albion Breeder Pak, Des Moines, IA), 108 g inorganic mineral (Inorganic Breeder Pak, Des Moines, IA, USA using the following sources: CaCO3 , CaHPO4 , MgO, MnO, MnS, CuSO4 , CuO, CoCO3 , ZnS, ZnO4 , KCl, NaCl), or no mineral daily for the 23 days prior to embryo collection. b Heifers received a daily supplement fed individually to each heifer containing mineral, soybean meal (SBM) and corn. c Heifers were allowed ad libitum access to hay. Intakes were determined by monitoring group intake for each treatment during a 7-day collection period. d Average daily individual mineral consumption based on total diet consumed by individual heifers.
On Days 12, 7, and 0, ovaries were scanned via transrectal ultrasonography (5-MHz intrarectal transducer, Aloka 500 V, Corometrics, Wallingford, CT) to determine the presence, number, and size of all follicles greater than 5 mm in diameter. The diameter of each follicle was calculated as the mean of the vertical and horizontal diameters. In addition, the presence and number of corpora lutea (CL) on each ovary were determined (Fig. 1). All ultrasonographic images were recorded on ovarian maps at the time of ultrasound for later review and analysis. Procedure MIXED was used to analyze noncategorical data (SAS Inst. Inc., Cary, NC). The model was used to analyze the total number of embryos collected, embryo stage and quality, degenerate embryos, unfertilized embryos, transferable embryos, follicles present on Day 7, CL present on Day 0, follicles from which ovulation had not occurred by Day 0, and the number of follicles determined on Day 7 for various size categories included treatment, period, square (replication in time), repetition (replication in space), heifer, and all two-way interactions with treatment. Heifer within treatment was used as the error term for period and square, whereas the residual error was used for repetitions and all interactions. All tests were determined to be significant when the P value for main effects was <0.05.
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Table 3 Ovarian follicle stimulation in heifers receiving inorganic, organic or no mineral after superovulation with follicle stimulating hormone Treatmentsa
Item
heifersb
All treated No. of heifers
P-value
Control
Inorganic
Organic
53
55
52
11.78 ± 1.11 7.97 ± 1.13 2.15 ± 0.53 0.66 ± 0.19
13.48 ± 1.09 8.21 ± 1.11 1.13 ± 0.52 0.59 ± 0.19
11.83 ± 1.09 6.97 ± 1.11 1.81 ± 0.52 0.55 ± 0.19
0.494 0.730 0.419 0.928
6.44 ± 0.89 1.75 ± 0.39
6.59 ± 0.87 1.99 ± 0.38
4.93 ± 0.87 1.74 ± 0.38
0.376 0.880
(n ± S.E.)
folliclesc
No. of >7 mm >9 mm >11 mm >13 mm
No. of corpora lutead No. of unovulated folliclese Responding heifersf No. of heifers No. of follicles >7 mm >9 mm >11 mm >13 mm No. of corpora lutead No. of unovulated folliclese
27
35
31
13.78 ± 1.46 9.76 ± 1.40 1.64 ± 0.76 0.50 ± 0.16
13.99 ± 1.20 8.75 ± 1.16 1.76 ± 0.62 0.77 ± 0.13
13.17 ± 1.35 7.09 ± 1.31 2.11 ± 0.71 0.56 ± 0.15
0.885 0.351 0.882 0.379
10.07 ± 1.13 2.45 ± 0.59
8.88 ± 0.94 2.69 ± 0.48
8.52 ± 1.06 2.85 ± 0.55
0.588 0.882
a Heifers received either 108 g of organic mineral, 108 g inorganic mineral, or no mineral for the 23 days prior to embryo collection. b All heifers receiving FSH (excluding 20 heifers from period five). c Determined 7 days before embryo collection by transrectal ultrasonography. d Determined on the day of embryo collection by transrectal ultrasonography. e Determined on the day of embryo collection by transrectal ultrasonography. f Only heifers responding to FSH (i.e., containing at least one CL at the time of embryo collection).
Overall, 180 embryo recoveries were scheduled (60 per treatment); however, 29 potential recoveries were removed from analysis associated with embryo numbers and quality because the recovery catheter was unable to be passed through the cervix or insufficient media was retrieved to warrant a successful recovery. In addition, ovarian ultrasound data during period five were not
Fig. 1. Timeline demonstrating superstimulation protocol for heifers assigned to inorganic, organic, or no mineral treatments. ECP, estradiol cypionate; FSH, follicle stimulating hormone; PGF, PGF2α ; CIDR, controlled internal drug release containing 1.9 g progesterone.
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collected because of damage to the ultrasonography machine. After initial analysis of all treated heifers, those that failed to respond to superstimulation of ovarian follicular development (defined as heifers that did not have at least one CL present at the time of embryo recovery on Day 0) were removed and a second analysis was performed using the same model. Pearson correlations were calculated among variables using the CORR procedure of SAS to determine correlations between: number of embryos recovered, degenerate embryos, unfertilized embryos, transferable embryos, and number and size class of follicles present on Day 7, CL present on Day 0, and unovulated follicles on Day 0. 3. Results Daily dry matter intake of hay was similar among treatments during the supplementation phase (Table 2). Among all heifers, daily dry matter intake was 14.1 ± 1.8 kg (mean ± S.D.). Of all the heifers assigned to a treatment, 63 69, and 56% of the control, inorganic, and organic treatment groups, respectively, had a CL on Day 12, when FSH was initiated. In addition, the response to superovulation was similar among mineral treatments (51, 64, and 60% for control, inorganic, and organic treatments, respectively). Data for response of heifers to superovulation treatments is summarized in Table 3. The number of follicles >7, >9, >11, and >13 mm on Day 7 was similar among all treatments. In addition, on Day 0 the number of CL and the number of follicles from which ovulation had not occurred was similar among all treatments. When heifers that failed to respond to superovulation treatments (i.e., there was no evidence of a CL on Day 0) were removed for separate analysis, the mean number of follicles on Day 7 and the number of CL on Day 0 increased, but no treatment differences were noted. Embryo number and quality data are summarized in Table 4. Among all heifers, the mean number of recovered embryos was similar among treatments (4.1 ± 0.7, 3.8 ± 0.7, and 3.3 ± 0.7 for control, inorganic-, and organic-treated heifers, respectively). The number of unfertilized oocytes was greater (P < 0.05) for inorganic (2.3 ± 0.4) and control (1.6 ± 0.4) groups than organictreated (0.4 ± 0.4) group. The number of degenerate and transferable embryos was similar among treatments. When data from heifers that failed to respond to superovulation were eliminated, the mean number of embryos increased to 6.5 ± 1.2 for control, 5.0 ± 1.0 for inorganic-treated, and 5.0 ± 1.1 for organic-treated heifers, respectively. Organic (0.5 ± 0.5) and inorganic (0.3 ± 0.4) treatment groups tended (P = 0.087) to have fewer degenerate embryos than control treated heifers (1.7 ± 0.5), whereas the number of unfertilized ova tended (P = 0.084) to be greater for inorganic (3.1 ± 0.7) than organic treatment (1.1 ± 0.7) groups. The number of unfertilized ova in the control (2.0 ± 0.8) group remained intermediate. As anticipated, there were differences (P < 0.001) among heifers in the total number of embryos recovered, which ranged from 0.3 ± 1.6 to 11.6 ± 1.9 embryos per recovery. Similarly, there were differences (P < 0.001) among heifers in the number of transferable embryos, unfertilized oocytes, and quality grade one and two embryos. In addition, there was a trend for a difference (P = 0.063) and a difference (P < 0.05) existed among heifers in the number of degenerate and quality grade three embryos, respectively. The total number of follicles >7 mm on Day 7 also differed (P < 0.001) among heifers, ranging from 5.4 ± 2.3 to 20.2 ± 2.3 follicles per recovery. At embryo recovery (Day 0), the average number of CL per heifer also differed (P < 0.001) and ranged from 0.7 ± 1.9 to 16.0 ± 1.9 CL per heifer. In addition, the number of follicles from which ovulation did not occur and were present on Day 0 also differed (P < 0.01) among heifers, ranging from 0.6 ± 0.8 to 4.4 ± 0.8 per heifer.
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Table 4 Embryo/ova production in heifers receiving inorganic, organic or no mineral after superovulation with follicle stimulating hormone Treatmentsa
Item
P-value
Control
Inorganic
Organic
(n ± S.E.) All treated No. of heifers No. of embryos/ova recovered No. of degenerate/cleaved No. of unfertilized No. of transferable Grade 1 Grade 2 Grade 3
49 4.09 ± 0.74 0.94 ± 0.33 1.56 ± 0.46c 1.61 ± 0.53 1.07 ± 0.44 0.52 ± 0.18 0.00 ± 0.04
51 3.79 ± 0.74 0.28 ± 0.33 2.34 ± 0.46c 1.14 ± 0.53 0.81 ± 0.44 0.31 ± 0.18 0.04 ± 0.04
51 3.29 ± 0.73 0.19 ± 0.31 0.44 ± 0.44c 2.13 ± 0.50 1.51 ± 0.42 0.58 ± 0.17 0.03 ± 0.04
0.462 0.266 0.024 0.445 0.555 0.569 0.550
Responding heifersd No. of heifers No. of mean embryos/ova recovered No. of degenerate/cleaved No. of unfertilized No. of transferable
23 6.54 ± 1.14 1.73 ± 0.49 1.97 ± 0.75 2.82 ± 0.79
31 4.99 ± 1.00 0.33 ± 0.43 3.10 ± 0.65 1.58 ± 0.69
30 4.99 ± 1.05 0.45 ± 0.46 1.12 ± 0.68 3.38 ± 0.73
0.549 0.087 0.084 0.150
heifersb
a
Heifers received either 108 g of organic mineral, 108 g inorganic mineral, or no mineral for the 23 days prior to embryo collection. b All heifers receiving FSH (excluding 29 heifers eliminated because of poor recovery of media). c Uncommon means within a row differ (P < 0.05) d Only heifers responding to FSH and when embryo retrieval was successful (i.e., containing at least one CL at the time of embryo collection).
Although there were no period × treatment interactions for any embryo characteristic variables, there were period effects during the study. The mean number of embryos collected per recovery per period did not differ among periods and ranged from 2.0 ± 0.9 to 5.4 ± 1.0 embryos per collection. Differences (P < 0.05) existed among periods in the number of unfertilized oocytes, transferable embryos, and quality grade two embryos. A tendency (P = 0.063) was noted for quality grade one embryos to differ among periods. The mean number of follicles >7 mm on Day 7 differed (P < 0.01) among periods and ranged from 7.8 ± 1.4 to 15.7 ± 1.4 follicles. The average number of CL on Day 0 was 5.8 ± 6.6 (mean ± S.D.) per period and was similar among periods; however, the average number of follicles from which ovulation did not occur ranged from 0.8 ± 0.5 to 3.8 ± 0.5 and was different (P < 0.05) among periods. Correlation coefficients between factors affected by superstimulation (number of follicles, size of follicles, and number of CL) and embryo collection factors (embryo number and quality) are summarized in Table 5. The most significant (P < 0.0001) correlation (r = 0.773) occurred between the number of CL at the day of embryo recovery and total number of collected embryos. However, the number of CL was also correlated with the number of degenerate, unfertilized oocytes, and transferable embryos (0.414; 0.428, and 0.422, respectively; P < 0.001). The number of follicles >7 mm on Day 7 had a greater correlation (r = 0.648; P < 0.0001) with total embryos recovered than the number of follicles >9, >11, or >13 mm on Day 7. Interestingly, number of follicles from which ovulation did not occur were correlated (r = 0.412; P < 0.0001) with the number of unfertilized ova.
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Table 5 Pearson correlation coefficients between the number of embryos for each embryo classification and ovarian follicle sizes, number of corpora lutea and follicles from which ovulation did not occur Embryo classificationb
Degenerate/cleaved Unfertilized Transferable Total ova/embryos a b c d e f g
Follicle sizesa >7 mm
>9 mm
>11 mm
>13 mm
0.288e
0.311f
0.480g 0.270e 0.648g
0.206 0.214e 0.426g
0.166 −0.088 0.194 0.148
−0.068 −0.046 −0.112 −0.139
Corpora luteac
Unovulated folliclesd
0.414f 0.428g 0.422g 0.773g
−0.071 0.412g −0.107 0.239
Determined seven days before embryo collection by transrectal ultrasonography. Classified according to the International Embryo Transfer Society (Savoy, IL). Determined on the day of embryo collection by transrectal ultrasonography. Determined on the day of embryo collection by transrectal ultrasonography. Correlation exists (P < 0.01). Correlation exists (P < 0.001). Correlation exists (P < 0.0001).
4. Discussion Current dogma in the embryo transfer community is that feeding organic mineral rather than inorganic mineral enhances the number of embryos and/or the quality of embryos recovered. Analysis of organic minerals has indicated that they may be of greater bioavailability compared with inorganic counterparts (Brown and Zeringue, 1994). Therefore, the potential exists for the enhanced bioavailability of organic trace minerals to enhance reproductive efficiency and potentially increase embryo number and quality. The organic product utilized in this study is a chelated (i.e., when a mineral is bound to amino acids with at least two bonds from each amino acid) mineral designed for donor cows in an embryo transfer program, with the recommendation that donors receive 108 g of the mineral daily for a minimum of 21 days prior to embryo recovery. The present study was designed to test the hypothesis that supplementing donor heifers with an organic mineral for three weeks prior to embryo recovery would enhance the number and quality of embryos collected in a superovulation program compared to those receiving an inorganic mineral or no mineral supplementation during the same period. As expected, not all heifers responded to superstimulation, but there was no difference among treatments in the percentage of heifers that did respond. It is well established that ovulation rate can be influenced by environmental factors such as nutrition and has become apparent that these nuritional effects are mediated by a direct action at the level of the ovary, involving insulin, insulin-like growth factors (IGF) I and II, and their binding proteins among other factors (Hunter et al., 2004). These factors can also affect the quality of the oocyte and consequently, embryo development and survival (Hunter et al., 2004). Bioavailability of organic minerals is greater than inorganic minerals and could potentially alter the nutrition-related factors affecting the number of oocytes available for ovulation or the quality of oocytes. However, in the current study mineral treatments started only 23 days prior to embryo collection. This may not have been sufficient time for the ovaries to be impacted by effects, if any, of mineral source to alter ovulation rates. Numerous studies have demonstrated nutritional effects on follicular and oocyte development and viability. Donors that had less than ideal energy intake prior to and during superovulation, had more follicles and improved embryo quality (Armstrong et al., 2001). Further, when oocytes were collected from hyperinsulinemic heifers oocyte quality was compromised (Adamiak et al., 2005)
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and sheep fed energy-restricted diets had increased embryo quality compared to high-energy diets (Nolan et al., 1998). Heifers fed a greater-energy diet were observed to have increased peripheral IGF-I and enhanced the intrafollicular IGF-I, resulting in increased growth rates of dominant follicles; however, cows receiving greater-protein diets had decreased oocyte quality, due in part to increased plasma urea concentrations (Armstrong et al., 2001). In the present study all heifers were maintained on a moderate energy and protein diet, which met but did not exceed NRC (1996) recommendations. The primary difference among treatments was trace mineral source. Perhaps these donors having been fed a well-balanced diet meeting recommendations for all nutrients negated the potential positive effects of feeding a more bioavailable mineral such as the organic mineral fed in the present study. We are unaware of data demonstrating local effects of trace mineral source on oocyte maturation or quality and follicular development. However, reports have indicated that the use of organic mineral has variable effects on reproductive performance in beef females. When beef cows were fed an organic mineral an increase in artificial insemination pregnancy rates during the subsequent breeding season were noted (Stanton et al., 2000). Further, young cows were noted to have increased reproductive performance after receiving organic mineral, whereas no effect was noted in mature cows (Arthington and Swenson, 2004). In sows, embryo and fetal survival were enhanced and incidence of return to estrus was reduced when sows received an organic mineral. In contrast, feeding supplemental organic or inorganic Cu did not enhance reproductive performance (Muehlenbein et al., 2001) and feeding excess organic or inorganic mineral reduced reproductive performance (Olson et al., 1999). These inconsistent data are similar with results from the present study. In the present study there was no difference in the mean number of embryos/ova recovered among the control, inorganic, or organic treatment groups, but there were subtle differences or tendencies for differences in the number of degenerate, unfertilized, and transferable embryos collected. Heifers receiving organic mineral had a tendency for a greater number of transferable embryos than inorganic treated heifers, but the increase appeared to be a result of an increase in the number of quality grade two embryos, rather than an increase in grade one embryos. However, no difference was detected between organic and control treatment groups. When eliminating the data for non-responding heifers, no differences in transferable embryos were detected. We hypothesized that feeding organic mineral would enhance embryo numbers. To increase embryo numbers, ovarian follicular dynamics and CL numbers would need to be altered to reflect the change in embryo numbers. Therefore, ultrasonographic examinations were performed to determine whether mineral treatment affected follicle development and ovulation rates. However, no differences were detected in follicle numbers, size of follicles, the percentage of follicles from which ovulation occurred, or the number of CL that developed demonstrating that organic or inorganic mineral supplementation during the 23 days prior to embryo recovery, did not appear to influence growth and ovulation from follicles. Even though all the heifers were half-siblings, specific heifers had greater embryo yields, more follicular growth, and ovulated from more follicles than other heifers. These data demonstrated that regardless of treatment, individual heifer had a greater impact on embryo quality and number than mineral treatment. In addition, period appeared to have a greater effect on embryo production than mineral treatment, primarily related to climatic differences among periods, which are known to have impacts on reproductive efficiency in cattle (de la Sota et al., 1998; Rensis and Scaramuzzi, 2003). The greatest correlation was demonstrated between number of embryos and the number of CL present on Day 0. Embryologists utilize palpation of CL at the time of recovery to predict total number of embryos and the correlation determined in this study (r = 0.773) was similar to
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that previously reported (Donaldson, 1985). However, an interesting observation of the current study was the number of unovulated follicles at the time of embryo recovery, which correlated (r = 0.412) to unfertilized oocytes. To our knowledge this is the first observation demonstrating this correlation and warrants additional research. Clearly a number of mechanisms are involved in mediating the effects of diet on ovarian function and the formulation of diets designed to optimize fertility in cattle must consider the effects of nutrient supply on follicular growth and oocyte quality. In the present study, heifers received a balanced diet to meet NRC (1996) recommendations and perhaps masked potential conclusive positive or negative effects of organic compared with inorganic mineral supplementation. However, heifer and period accounted for the greatest differences in embryo production and quality. Following 23 days of mineral supplementation, which was the feeding protocol used in this experiment, neither organic nor inorganic minerals appeared to significantly alter embryo numbers and quality. Acknowledgements Appreciation is expressed to John Mundhenke, Mundhenke Beef, Lewis, KS for use of heifers to conduct this study. The authors thank Albion Advanced Nutrition, Clearfield, UT for partial financial support and donation of mineral products and Pfizer Animal Health, New York, NY, for donation of PGF2α (Lutalyse® ) and CIDR inserts. Appreciation also is expressed to S. Logan, A. Perez-Salazar, A. Spell, K. Thielen, and R. Wasson for their assistance with data collection. References AAFCO. 2000. Official Publication. Association of American Feed Control Officials, Inc., Oxford, IN. Adamiak, S.J., Mackie, K., Watt, R.G., Webb, R., Sinclair, K.D., 2005. Impact of nutrition on oocyte quality: cumulative effects of body composition and diet leading to hyperinsulinemia in cattle. Biol. Reprod. 73, 918–926. Armstrong, D.G., McEvoy, T.G., Baxter, G., Robinson, J.J., Hogg, C.O., Woad, K.J., Webb, R., Sinclair, K.D., 2001. Effect of dietary energy and protein on bovine follicular dynamics and embryo production in vitro: associations with the ovarian insulin-like growth factor system. Biol. Reprod. 64, 1624–1632. Arthington, J.D., Swenson, C.K., 2004. Effects of trace mineral source and feeding method on the productivity of grazing Braford cows. Prof. Anim. Sci. 20, 155–161. Baily, J.D., Ansotegui, R.P., Paterson, J.A., Swenson, C.K., Johnson, A.B., 2001. Effects of supplementing combinations of inorganic and complexed copper on performance and liver mineral status of beef heifers consuming antagonists. J. Anim. Sci. 79, 2926–2934. Brown, T.F., Zeringue, L.K., 1994. Laboratory evaluations of solubility and structural integrity of complexed and chelated trace mineral supplements. J. Dairy Sci. 77, 181–189. de la Sota, R.L., Burke, J.M., Risko, C.A., Moreira, F., DeLorenzo, M.A., Thatcher, W.W., 1998. Evaluation of timed insemination during summer heat stress in lactating dairy cattle. Theriogenology 49, 761–770. Donaldson, L.E., 1985. Estimation of superovulation response in donor cows. Vet. Rec. 117, 33–34. Graham, T.W., 1991. Element deficiencies in cattle. Vet. Clin. North Am. Food Anim. Pract. 7, 153–215. Hunter, M.G., Robinson, R.S., Mann, G.E., Webb, R., 2004. Endocrine and paracrine control of follicular development and ovulation rate in farm species. Anim. Reprod. Sci. 82–83, 461–477. Muehlenbein, E.L., Brink, D.R., Deutcher, G.H., Carlson, M.P., Johnson, A.B., 2001. Effects of inorganic and organic copper supplemented to first-calf cows on cow reproduction and calf health and performance. J. Anim. Sci. 79, 1650–1659. Nolan, R., O’Callaghan, D., Duby, R.T., Lonergan, P., Boland, M.P., 1998. The influence of short-term nutrient changes on follicle growth and embryo production following superovulation in beef heifers. Theriogenology 50, 1263–1274. Olson, P.A., Brink, D.R., Hickok, D.T., Carlson, M.P., Schneider, N.R., Deutscher, G.H., Adams, D.C., Colburn, D.J., Johnson, A.B., 1999. Effects of supplementation of organic and inorganic combinations of copper, cobalt, manganese, and zinc above nutrient requirement levels on postpartum two-year-old cows. J. Anim. Sci. 77, 522–532.
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