1. Introduction
Forage is the primary component of beef cows’ diets; however, forage often does not provide adequate amounts of trace minerals; therefore, mineral supplementation is often required [
1]. Copper and zinc are widely deficient in forages across the U.S., and the selenium content of forages is quite variable with some locations having extreme deficiencies and others being sufficient [
2]. The trace mineral status of cows at calving can affect the calf’s mineral status at birth and its health in early life [
3]. Additionally, a cow’s reproductive performance can be impacted by her trace mineral status [
4]. Specifically, the trace minerals Cu, Se, and Zn play important roles in reproduction [
3].
The most common form of mineral supplementation in grazing systems is free-choice mineral [
5]. Typically, a cow’s appetite for a free-choice mineral is not related to her mineral requirement, but rather to her taste for salt, thus the intake of free-choice mineral and, subsequently, the mineral status of cows within a herd can be variable [
6]. Injectable trace minerals can be used to provide additional supplemental trace minerals and allow for a known amount of trace minerals to be delivered to each animal. Trace mineral injections have been proven to increase the status of animals in the short term [
7,
8].
However, little research has been conducted to evaluate the effects of supplementation of trace minerals through an injection in cow–calf production systems when free-choice mineral is provided. Mundell et al. [
9] reported improved conception to fixed-time artificial insemination in beef cows given trace mineral injections 105 days before calving and again 30 days before breeding, when grazing native range in Kansas, USA. However, after exposure to clean-up bulls, no difference in overall pregnancy rate was detected. The calves nursing injected dams also received trace mineral injections at birth and at approximately 71 days of age, but no improvements in their growth were observed. However, in Illinois, USA, Stokes et al. [
10] reported a reduced fixed-time artificial insemination conception rate of first-calf heifers, receiving repeated trace mineral injections, with no differences in overall pregnancy rate after exposure to clean-up bulls. The heifers receiving the trace mineral injection had greater milk production, and their calves had greater Cu and Se status at birth, but no differences in calf growth rates were observed.
The objective of this trial was to determine the effects of a trace mineral injection on the trace mineral status of cows and calves and the resulting impacts on the reproductive performance of the cows and the growth of their nursing calves.
2. Materials and Methods
All procedures and protocols were approved by the University of Idaho Institutional Animal Care and Use Committee.
Crossbred beef cows (Angus, Hereford, and Angus × Hereford cross) and their calves located at the Nancy M. Cummings Research Extension and Education Center in Carmen, ID, USA, were used in this study. The trial was initiated in December of 2012. During the summer prior to the initiation of the study, approximately half of the 200 cows used in this study grazed native range, while the remainder were maintained on irrigated pasture. Cows were stratified by their summer grazing regimen, age (range from 2 to 13 years with 24% being 2 to 4, 66% being 5 to 10, and 10% being 11 to 13 years old), and expected calving date and assigned to receive trace mineral injections (TMI) of Multimin 90 (Multimin, Fort Collins, CO, USA) containing 15 mg/mL Cu, 10 mg/mL Mn, 5 mg/mL Se, and 60 mg/mL Zn at pre-calving and pre-breeding or to remain as untreated controls (CON). The cow was considered the experimental unit, and cows remained on the same treatment over the two-year trial.
During the winter and early spring periods (December–May), cows were fed alfalfa hay and wheat straw. In May, the cows began grazing on irrigated orchard grass (
Dactylis glomerata) pastures and continued grazing these pastures until December. During this time, cows were rotationally grazed on pasture that were approximately 6 ha each. The trace mineral concentration of these forages (
Table 1) was analyzed by a commercial laboratory (Cumberland Valley Analytical Services, Hagerstown, MD, USA) using the Metals and Other Elements in Plants protocol [
11]. Modifications to the protocol included ashing a 0.35 g sample for 1 h at 535 °C and digesting in open crucibles for 20 min in 15% nitric acid on hotplates. Samples were then diluted to 50 mL and analyzed by inductively coupled plasma spectroscopy.
All forages were deficient in Cu, Se, and Zn. Additionally, the pasture contained moderately antagonistic concentrations of molybdenum. Manganese was adequate in the pasture and would have been marginal in the winter alfalfa hay/straw diet. A custom-made free-choice mineral supplement (
Table 2) was offered throughout the entirety of the trial with the exception of a 53-day period in year 1 (32–85 days post artificial insemination) when they were provided with a commercial free-choice mineral supplement (Purina Wind and Rain Storm All Season 7.5 Complete) due to the custom mineral being unavailable. Salt was added to the mineral mixes to achieve a targeted intake of 113 g/cow/day of the mineral mix.
2.1. Cow Management Timeline
An overview of events during the trial is shown in
Figure 1. In December of 2012 (50 days prior to the start of calving season), cows in the TMI group were injected with 6 mL of Multimin 90 (0.67 mL/68 kg of body weight). Then, at 23 days prior to AI (117 d post pre-calving injection), cows in the TMI group were again given 6 mL of Multimin 90 (0.69 mL/68 kg of BW). All cows were estrus synchronized using a 5-day CO-Synch plus CIDR protocol, in which cows were given a 2 mL injection of gonadotropin releasing hormone (GnRH, 43 μg/mL; Fertagyl, Merck Animal Health) and received an insert of a controlled internal drug release device (CIDR vaginal insert containing 1.38 g of progesterone; Eazi-Breed CIDR, Zoetis Animal Health, New York, NY, USA) 8 days prior to AI (day 132). The CIDR was removed 5 days later (day 137) and an injection of prostaglandin F
2α (PG; 25 mg; Lutalyse, Zoetis Animal Health) followed by a second PG injection 5.6 h later was administered. Cows were administered an injection of GnRH and inseminated either 72 or 80 h after CIDR removal (day 140) with sexed semen. Cows were stratified across injection treatment to one of the two insemination times (72 or 80 h). Cows were randomly inseminated by one of two technicians and then exposed to fertile bulls for natural-service breeding 17 days after AI (day 158) and remained with the bulls for 45 days. Pregnancy was determined using ultrasonography at 55 days post AI (day 195) and by rectal palpation at 105 days post AI (day 245). The artificial insemination pregnancy rate was calculated using ((n pregnant to AI/total
n synchronized) × 100).
Some cows (n = 14 CON and n = 12 TMI) were removed from the study during year 1 due to being culled from the herd for loss of calf (n = 1 CON and n = 2 TMI) or other management reasons (behavior or conformation). Additionally, cows were culled at pregnancy check in October for failing to conceive. Bred heifers that had received a TMI (n = 11) or not (n = 10) prior to breeding as heifers in year 1 (but were not a part of this study in year 1) were added to the trial in year 2 starting at the time of the pre-calving injection (day 365; December 2013) to replace cows removed from the study.
In December of 2013 (36 days prior to the start of calving season; day 365), cows in the TMI group were injected with 7.5 mL of Multimin 90 (0.75 ± 0.014 mL/68 kg of BW). Twenty-eight days prior to AI (day 401), cows in the TMI group were given 6.5 mL of Multimin90 (0.75 ± 0.020 mL/68 kg of BW). Cows were estrus synchronized using a modification of the 5-day CO-Synch plus CIDR protocol. All estrous synchronization products used were the same as in Year 1. Briefly, cows received an injection of GnRH and a CIDR insert on day 500. The CIDR was removed 5 days later (day 505), an injection of PG was administered, and an estrus detection aid (EstroTect, EstroTect, Inc. Denver, CO, USA) was applied. Cows with activated detection aids (50% or greater activation) were then inseminated by fixed-time AI at 72 h with non-sexed (n = 72 CON and 65 TMI) or sexed semen (n = 22 CON and 19 TMI) by one of three technicians (days 508 through 509). Cows that showed no estrous response to synchronization by 72 h post CIDR removal were inseminated by fixed-time AI at 96 h post CIDR removal. Regardless of estrous response, all cows received GnRH at fixed-time AI. Cows were exposed to fertile bulls on day 518 and co-mingled for 49 days. Pregnancy was determined using ultrasonography at 56 days post AI (day 567) and by rectal palpation at 150 days post AI (day 659). Cows that lost calves between calving and breeding in year 2 were culled from the herd (n = 3 CON and n = 2 TMI). Additionally, cows that were less than 30 days post-calving at the start of the breeding (n = 5 CON and n = 7 TMI) were not synchronized and were removed from the pregnancy analysis in year 2. To evaluate the potential effect of TMI on timing of conception, change in calving date in year 2 was calculated by subtracting 365 from the number of days between calving in year 1 and calving in year 2.
All cows were evaluated for body condition score (BCS; 1—emaciated, 9—obese), before the start of calving (December), one month prior to the start of breeding (April), and at summer pregnancy check (July).
2.2. Calf Management Timeline
Calves were sired by Angus, Hereford, or Simmental bulls, with calves from TMI cows receiving a 1 mL injection of Multimin 90 between birth and 24 h of age in both years. In year 1, TMI calves also received an injection at branding on day 119 of the trial (1 mL/45 kg BW, 49 ± 1.3 days of age (DOA)). In year 1, jugular blood was collected from calves (
n = 40, 20 per treatment) at branding to determine plasma trace mineral concentrations. The weight of calves was recorded at birth (days 50–110 and days 401–461) branding (day 119; 49 ± 1.3 DOA, year 1 only), summer pregnancy check (days 195 and 567; 128 ± 1.3 DOA, year 1; 128 ± 1.2 DOA, year 2), and at weaning (days 265 and 632; 197 ± 1.3 DOA, year 1; 193 ± 1.2 DOA, year 2). Two-hundred and five day adjusted weaning weights were calculated using the Beef Improvement Federation guidelines [
12].
Calves were removed from analysis if they died (n = 5 CON and 6 TMI in year 1; 2 CON, and 3 TMI in year 2) or were born as twins (4 and 3 sets of twins for CON and TMI, respectively in year 1). Thus, 91 CON and 91 TMI calves were used to evaluate the effects of TMI in year 1 and 98 CON and 97 TMI calves were used in year 2.
2.3. Liver Sampling
Liver biopsies were collected from 20 cows per treatment that were selected randomly. Cows were sampled at pre-calving (days 0 and d 365), pre-breeding (days 117 and 478), 15 to 18 days post pre-breeding TMI (referred to as breeding; days 132 and 496), and near weaning (days 293 and 632) to determine liver trace mineral concentrations. Biopsies were collected from the same cows at each sampling date. Liver biopsies were also collected from the calves (
n = 40, 20 CON, 20 TMI) of sampler cows at weaning (days 265 and 632). Liver biopsies were collected using the method of Engle and Spears [
13]. Liver biopsy samples were placed in a plastic culture tube, transported on ice to the laboratory, and frozen at −20 °C. Liver samples were then dried in a forced-air oven (60 °C). Samples were then shipped to the Diagnostic Center for Population and Animal Health (Lansing, MI, USA) and analyzed for trace mineral concentration. Once at the laboratory, tissues were dried overnight in a 75 °C oven and then digested overnight in nitric acid. Elemental analysis followed the methods of Wahlen et al. [
14] using an Agilent 7500ce Inductively Coupled Plasma–Mass Spectrometer (Agilent Technologies Inc., Santa Clara, CA 95051, USA). Elemental concentrations were calibrated using a four-point liver curve of the analyte–internal standard response ratio. The lowest concentrations points were 0.1 µg/mL for Cu and Zn, 0.5 ng/mL for Mn, and 0.1 ng/mL for Se. Standards were from GFS (GFS Chemicals, Powell, OH 46065, USA). A National Institute of Standards and Technology (NIST, Gaithersburg, MD 20899, USA) bovine liver standard was used as a control.
2.4. Statistical Analysis
For all models, the individual animal was the experimental unit and significance was declared at p ≤ 0.05. Due to the lower number of samples and, thus, power, tendencies are discussed when 0.05 < p ≤ 0.15 for all mineral analysis.
Liver mineral concentrations of Cu, Mn, Se, and Zn were analyzed using the mixed procedure of SAS (PROC MIXED; SAS Inst., Inc., Cary, NC, USA) with the fixed effect of treatment, day, and their interaction included in the models. Day was considered a repeated effect. The covariates of initial pre-treatment liver concentrations (day 0) were used as a covariate in all models.
Cow BCSs were analyzed using a mixed-effects model with treatment and day as fixed effects. The treatment by day interaction was not significant (p = 0.81) and was removed from the model. Day was considered a repeated effect. Cow BCS recorded on day 0 was used as a covariate.
Pregnancy rates were analyzed using logistic regression (PROC GENMOD; SAS Inst., Inc., Cary, NC, USA). For year 1, the model used to assess differences in AI pregnancy rate tested the effects of treatment and AI timing treatment (72 vs. 80 h) and their interaction. The interaction was not significant (p = 0.54) and was removed from the model. The AI timing was not significant (p = 0.37) and was removed from the model. The covariates of AI sire and AI technician were tested. The covariate of AI technician was not significant (p = 0.19) and was removed from model. The model used to assess differences in overall pregnancy rate included the fixed effect of treatment.
For year 2, the model used to assess differences in AI pregnancy rate included treatment, semen type (sexed vs. non-sexed), and timing of AI (heat response (72 h) vs. delayed 96 h after CIDR removal) and their interactions. There were no significant interactions (p > 0.42) and all interactions were removed from the model. The fixed effects of semen type and timing of AI were not significant (p ≥ 0.31) and were removed from the model. The covariates of AI sire and AI technician were tested. The covariate of AI sire was not significant (p = 0.47) and was removed from the model.
Calf plasma concentrations of Cu, Mn, Se, and Zn, in year 1, were analyzed using the mixed procedure of SAS, and treatment was considered a fixed effect with calf sex being used as a covariate.
Within each year, calf birth weight, average daily gain (ADG), weaning weight, 205-day adjusted weaning weight, and liver mineral concentrations were analyzed using a mixed-effects model including the fixed effect of treatment. Calf sex and dam age were used as covariates for calf birth weight, ADG, and actual weaning weight. Dam age and calf sex were used as covariates for liver mineral concentrations.