Effects of dietary Zn on ewe milk minerals and somatic cell count

INTRODUCTION

Gestating and lactating ewes require nutritional management with adequate provision of macro- and micronutrients. Protein and energy requirements increase from midgestation to early lactation but less information is available on the importance of Zn during these physiologically demanding production time points in sheep. Furthermore, the amount of Zn in dormant forages may be insufficient to meet animal requirements at these time points in range- or pasture-based production systems (Sprinkle et al., 2018). Blood minerals were altered during clinical mastitis, and have also been shown to be lower in cases of subclinical mastitis in dairy cattle (Naresh et al., 2001). Considering the role of Zn in immune function and inflammation, it is possible that Zn sequestered in mammary secretions could be used to aid in countering bacterial challenges (Suttle, 2010; Bonaventura et al., 2015), indicating potential benefits from supplementing increased dietary Zn prior to parturition and during lactation.

Therefore, the objectives of the current study were to evaluate the effects of increased dietary Zn fed at approximately three times the NRC recommendation (NRC, 2007) from late gestation to early lactation on milk Zn and mammary health by quantifying milk somatic cell count (SCC). We hypothesized that increased dietary Zn fed above NRC recommendations would increase milk Zn and positively affect mammary health as indicated by SCC.

MATERIALS AND METHODS

The experimental protocol used in this study was approved by the Institutional Animal Care and Use Committee at the University of Wyoming (#A-3216-01).

Study Design and Treatment Diets

The experiment utilized primiparous Rambouillet (WF; n = 48) and Hampshire (BF; n = 41) type ewes ranked by weight (77 ± 6 kg) and breed type. Ewes were then randomly assigned along the ranking to one of two supplement treatment groups: Control (CON; n = 45, 37 mg Zn/kg Dry Matter (DM), ≈1× NRC) and Zn treatment (ZnTRT; n = 44, 113 mg Zn/kg DM, ≈3× NRC). Ewes from each treatment group were managed together on pasture and each received 0.45 kg of a ZnSO₄-fortified alfalfa pellet corresponding with their designated dietary Zn treatment (Table 1) for approximately 6 wk prior to parturition and for 30 d postlambing. Ewe supplement intake was controlled and measured by a Super Smartfeeder (C-Lock Inc., Rapid City, SD), which quantified and allotted a maximum of the 0.45 kg/d of supplement by reading each ewe’s electronic identification tag before dispensing their corresponding treatment. Use of this technology allowed ewe to be the experimental unit. Ewes were adapted to the feeder for 10 d prior to when treatments were initiated. Dietary treatments started on November 6, 2018 (d 0) on dormant meadow brome (Bromus riparius) and timothy (Phleum pretense) pasture. On d 50 (≈d 123 of gestation) of supplementation, ewes were moved in preparation for lambing into drop pens (40 × 20 m) in a three-sided barn where the SmartFeeder was fenced between two pens and treatment groups were divided to ensure no nontarget consumption occurred across treatment groups during confinement. Ewes had ad libitum access to water and grass hay (19.6 mg/kg Zn; Table 1), while in the drop pens and for the 30 d postlambing in group pens.

Table 1.

Chemical and nutrient composition of feedstuffs and treatment pellets fed to yearling ewes

Nutrient composition1	Dormant pasture	Grass hay	Treatment pellet
			CON	Zn treatment
Dry matter, %	90.26	88.65	92.16	92.98
Crude protein, %	3.6	6.1	19.7	19.6
Metabolizable, Mcal/kg	1.78	2.10	2.11	2.12
Acid detergent fiber, %	46.6	38.9	34.1	33.7
Total digestible nutrients, %	49.4	58.1	58.5	58.8
Mineral composition
Ca, %	0.52	0.39	1.19	1.28
P, %	0.06	0.17	0.26	0.26
K, %	0.56	1.92	3.03	3.18
S, %	0.07	0.19	0.29	0.32
Mg, %	0.15	0.16	0.28	0.29
Na, %	0.04	0.21	0.11	0.12
Fe, mg/kg	117	68	181	186
Mn, mg/kg	51	246	91	112
Cu, mg/kg	2.2	5.1	10.6	10.5
Zn, mg/kg	6.5	19.6	82.3	475.3
Mo, mg/kg	0.61	1.88	3.52	4.05

¹Analyzed by Ward Laboratories, Inc. (Kearney, NE).

Milk Sampling and Analysis

Milk samples were collected twice weekly beginning at 18 ± 6 h postlambing until approximately 30 d postlambing. Teats were cleaned prior to milk collection using an ethanol (70%) wipe. Approximately 20 mL of milk was collected from each udder half and combined into a single 50 mL conical tube. Samples were kept on ice until SCC was quantified within 24 h of milk collection using a somatic cell counter (LactiCyte HD, Page & Pedersen International, Ltd, Hopkinton, MA). After quantifying SCC, milk was stored and frozen at −20 °C until mineral element analysis. Frozen milk samples collected at approximately d 0, 10, and 30 of lactation were analyzed for mineral concentrations using dry digestion and ionized coupled plasma mass spectrometry (Wahlen et al., 2005).

Statistical Analysis

Milk mineral element concentrations were analyzed as repeated measures in the MIXED procedure of SAS (v9.4; SAS Inst. Inc., Cary, NC) with fixed effects of treatment (CON and ZnTRT), breed type (BF or WF), litter size (1 or 2+), day of lactation (d 0, 10, or 30), and all two-way interactions. Ewe SCC was log transformed (LogSCC) and analyzed with the same effects but with more levels for day of lactation (d 3 to 5, 6 to 9, 10 to 12, 13 to 16, 17 to 19, 20 to 23, 24 to 26, 27 to 29, or 30 to 32). Autoregressive type 1 (milk mineral) and Toeplitz (LogSCC) covariance structures were determined to be the most parsimonious by Akaike’s Information Criterion.

RESULTS

There was a breed × day of lactation interaction for milk Zn concentration (P = 0.04) due to a numerical reranking but not significant difference between breeds across day of lactation (P ≥ 0.06). Least-squares means for the main effects on milk mineral concentrations are displayed in Table 2. As a main effect, breed did not influence milk Ca, Cu, Mg, P, or Zn (P ≥ 0.10). Single-bearing ewes had greater milk Ca, Mg, and P than multiple-bearing ewes (P ≤ 0.04). Lactation day influenced milk Mg, P, and Zn and values generally decreased as lactation progressed (P < 0.01). Milk Zn was greater for ZnTRT than CON (P < 0.01), but Ca, Mg, and P concentrations were all greater for Control than Zn treatment (P ≤ 0.02).

Table 2.

Least-squares means (±SE) for the main effects of ewe treatment, breed, litter size, and lactation period on milk mineral concentrations

Effect	Level	Mineral
		Ca, g/L	Cu, mg/L	Mg, g/L	P, g/L	Zn, mg/L
Treatment1	CON	2.29 ± 0.07	2.56 ± 0.35	0.31 ± 0.01a	2.41 ± 0.08a	6.10 ± 0.78b
	ZnTRT	2.05 ± 0.07	1.79 ± 0.34	0.28 ± 0.01b	2.12 ± 0.07b	10.57 ± 0.79a
Breed	BF	2.20 ± 0.07	2.43 ± 0.34	0.29 ± 0.01	2.36 ± 0.07	8.49 ± 0.79
	WF	2.14 ± 0.07	1.92 ± 0.35	0.30 ± 0.01	2.18 ± 0.08	8.19 ± 0.80
Litter size	1	2.29 ± 0.07a	2.52 ± 0.35	0.31 ± 0.01a	2.4 ± 0.08a	9.13 ± 0.80
	2+	2.05 ± 0.07b	1.83 ± 0.34	0.28 ± 0.01b	2.13 ± 0.07b	7.55 ± 0.78
Lactation period, d	d 0	2.10 ± 0.08	2.57 ± 0.41	0.33 ± 0.01a	2.63 ± 0.09a	12.62 ± 0.95a
	d 10	2.24 ± 0.08	1.81 ± 0.42	0.27 ± 0.01b	2.14 ± 0.09b	7.89 ± 0.97b
	d 30	2.17 ± 0.08	2.14 ± 0.41	0.29 ± 0.01a,b	2.03 ± 0.09b	4.5 ± 0.95c

¹CON = 37 mg Zn/kg DM and ZnTRT = 113 mg Zn/kg DM; BF = Hampshire ewes, WF = Rambouillet ewes; 1 = single-bearing; 2+ = multiple-bearing.

^a,b,cMeans within an effect with no common superscript are different (P < 0.04).

A breed × litter size effect was detected for LogSCC (P = 0.02). Single-bearing WF ewes had lower LogSCC than multiple-bearing WF ewes (5.36 ± 0.09 vs. 5.74 ± 0.07; P < 0.01) but litter size did not affect BF ewe LogSCC (5.80 ± 0.08 vs. 5.79 ± 0.09; P = 0.92). Least-squares means for main effects of treatment and lactation period on ewe LogSCC are presented in Table 3. Ewe LogSCC was affected by day of lactation (P < 0.01) and was greatest within the first 9 d of lactation. However, CON and ZnTRT ewes did not differ in LogSCC (P = 0.25).

Table 3.

Least-squares means (±SE) for the main effects of ewe treatment and lactation period on LogSCC

Effect	Level	LogSCC
Treatment1	CON	5.72 ± 0.06
	ZnTRT	5.62 ± 0.06
Lactation period, d	3–5	5.93 ± 0.06a
	6–9	5.94 ± 0.06a
	10–12	5.77 ± 0.06a,b
	13–16	5.58 ± 0.06c
	17–19	5.56 ± 0.06c
	20–23	5.55 ± 0.06c
	24–26	5.56 ± 0.06c
	27–29	5.59 ± 0.06b,c
	30–32	5.54 ± 0.06c

¹CON = 37 mg Zn/kg DM and ZnTRT = 113 mg Zn/kg DM.

^a,b,cMeans within an effect with no common superscript are different (P < 0.04).

DISCUSSION

Feeding increased dietary Zn concentrations to dairy cattle can increase milk Zn concentrations and help to reduce SCC, which may reduce the incidence of clinical and subclinical mastitis (Pechová et al., 2006; Sobhanirad et al., 2010). This is thought to be in part due to the role of Zn in the formation of the keratin lining of the teat and protection it offers from bacterial infection and the extensive influence of Zn in immunity function and inflammation (Spain, 1994; Suttle, 2010).

Murphy et al. (2018) reported a 26% decrease in serum Zn concentrations in ewes with greater milk SCC, where ewes with greater SCC (>500,000/mL) weaned between 6.4 and 22.2 kg less lamb than ewes with less milk SCC (<500,000/mL). While these findings in U.S. range sheep may give novel insight on often inconsistent negative production impacts of subclinical mastitis, the impacts of clinical mastitis in sheep production systems has been well documented, including decreased milk production, reduced Average Daily Gain in lambs, and even increased mortality (Watson and Buswell, 1984; Leitner et al., 2008). The current research is consistent with previous estimates that susceptibility to intramammary infection is generally greatest around the time of parturition, with SCC peaking in the first 9 d postparturition (Waage and Vatn, 2008). These findings provide valuable information regarding peak SCC to guide preventative or therapeutic intervention strategies in shed-lambing systems in the United States.

In the current study, ewe milk Zn increased 73% compared with CON when dietary Zn was three times NRC recommendations (NRC, 2007). These findings are consistent with results in dairy cattle fed 8.5 times NRC recommendations (NRC, 2001; Sobhanirad et al., 2010). However, results from the current study are different from similar research in dairy cattle that saw no difference in milk Zn when supplemented chelated Zn at approximately four times NRC recommendations (Pechová et al., 2006). The ability to increase milk Zn through increased dietary Zn is intriguing due to the fact that biologically available Zn is regulated with various transport mechanisms and is maintained within a narrow range (Suttle, 2010).

Milk Zn may be increased due to transport stimulation in the mammary gland by glucocorticoids at and around parturition when glucocorticoid levels are high, potentially explaining why milk Zn concentrations were highest at lambing in the current study, and decreased as lactation progressed. Vaillancourt and Allen (1991) reported the greatest milk Zn in cow colostrum in the first day after parturition followed by a significant decrease of approximately 70% from h 12 to 72 postparturition. Furthermore, milk Zn was not different from d 3 to d 150 of lactation.

Dietary Zn had no effect on SCC in the current study, which is consistent with observations in dairy goats supplemented Zn methionine at seven times NRC requirements (Spain, 1994; Salama et al., 2003). In combination with results from the current study, these suggest current NRC recommendations may be adequate independent of Zn source for small ruminant mammary health. However, Pechová et al. (2006) reported a 45% decrease in SCC among dairy cows fed 245 mg/kg Zn (≈4× NRC requirements) compared with their control (190 mg/kg Zn; ≈3× NRC requirements). The effects of dietary Zn concentrations and Zn source on specific mastitis causing bacteria are not well understood and warrants additional research as the current study utilized ZnSO₄. Rambouillet SCC was greater in multiple- than single-bearing ewes but Hampshire ewe SCC was unaffected by litter size in the present study. Waage and Vatn (2008) observed incidence of clinical mastitis increased with multiple lambs born per ewe, with incidence of clinical mastitis in twin-bearing ewes approximately three times more than single-bearing ewes.

We demonstrated that increased dietary Zn results in increased milk Zn in ewes fed above NRC recommendations in late gestation and early lactation. The effects of increased milk Zn concentrations on neonate performance, passive immunity, and lamb serum Zn concentrations are concurrently being evaluated. Feeding Zn at three times NRC recommendations had no effect on SCC in a simulated western shed-lambing production system like the current study. Still, results provide longitudinal values of SCC throughout lactation which may inform preventative intervention strategies for cases of subclinical mastitis since peak SCC is within the first 9 d postlambing.

Conflict of interest statement. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture (USDA). The USDA is an equal opportunity provider and employer. The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.