Increasing vitamin D levels to improve fertilization rates in cattle

Abstract

Recently, interest in supplementing vitamin D (Vit D) to improve aspects of health, mainly in human fertility, has emerged. Still, supplementation of Vit D above the minimum required levels has yet to be explored in cattle despite evidence for Vit D receptors in reproductive tissues. The objective of this study was to establish if a dose-response relationship exists between Vit D exposure and success of in vitro production (IVP) of embryos and, if acute supplementation of Vit D improves pregnancy rates during timed artificial insemination (TAI) of dairy cows. Cumulus-oocyte complexes (COCs) were obtained from ovaries acquired from a local abattoir and cultured in five different IVP treatments from three separate collections (Control, 50, 100, 150, and 200 ng/mL of 1,25(OH)2D3; n = 20-30 COCs/group). In Experiment 2, dairy breed cows (n = 100) were synchronized for TAI with the PresynchOvsynch protocol. Cows received 150,000 IU of Vit D (n = 48) or castor oil as control (n = 53) along with gonadotropin-releasing hormone (GnRH) 24 h before TAI. Serum samples were collected before and 24 h after treatment. A small cohort of cows (n = 4) received the same treatments in two separate cycles and follicular fluid (FF) was collected after 24 h for calcidiol (25OHD) analyses. Increased concentrations of Vit D resulted in decreased rates of maturation of COC (150 and 200 ng/mL vs. control and 50 ng/mL; P = 0.01). Supplementation with 50 ng/mL resulted in greater numbers of early blastocyst and blastocyst stage embryos (P < 0.009). Pregnancy at first breeding did not differ (P = 0.13) between groups, but serum 25OHD increased in treated females after 24 h (P = 0.002). The FF 25OHD levels were reflective of serum levels, however, the observed increase in the treatment cycle (P = 0.04) was parallel to an overall increase in serum 25OHD during the entire second cycle, likely due to increased environmental sunlight exposure (March, control vs. May, treatment). A similar increase in the serum 25OHD in the lactating commercial herd maintained in covered housing was not observed, although experiments were conducted during a similar timeframe. This herd had levels of 25OHD near the low end of sufficiency according to National Research Council (NRC) guidelines. We conclude mild Vitamin D supplementation with concentrations at the higher end of NRC guidelines can improve maturation rates of recovered COCs. However, longer term supplementation may be needed to appreciate any benefits on fertility.

Keywords: Vitamin D; cattle; embryo7fertility; fertilization.

Figure 1.

Timeline of treatments to determine if exogenously administered Vitamin D₃ supplement increases ovarian/follicular 25OHD concentrations within 24 h. Non-lactating Jersey breed adult cows (n = 4) were subjected to two sequential treatments in three successive estrous cycles in a Pre-synch/Ov-synch estrus synchronization protocol from March to May in Corvallis, OR, USA. The standard Pre-synch Ovsynch protocol is depicted with the series of PGF_2α, and GnRH injections to synchronize ovulation. Females first received the vehicle control on day 34; after an estrous cycle off, females underwent a subsequent synchronization cycle and received Vitamin AD injectable (150 kIU Vitamin D₃, 1,000 kIU Vitamin A). Blood was collected immediately prior to treatments on day 34 (Day −1) and again 24 h later (day 0). Follicular fluid was collected 24 h after treatment with vehicle or Vitamin AD injectable (Created with Biorender.com).

Figure 2.

Timeline of treatments to determine if exogenously administered Vitamin D₃ supplement 24 h prior to timed artificial insemination (TAI) improves pregnancy rates. Lactating Holstein breed adult cows were subjected to treatments in a Pre-synch/Ov-synch estrus synchronization protocol from April to June in Monroe, OR, USA. The standard Pre-synch Ovsynch protocol is depicted with the series of PGF_2α, and GnRH injections to synchronize ovulation. Females either received the vehicle control on day 34 (n = 54), or Vitamin AD injectable (150 kIU Vitamin D₃, 1,000 kIU Vitamin A; n = 51). Blood was collected immediately prior to treatments on day 34 (day −1) and again 24 h later (day 0). Females were bred by artificial insemination (AI) 24 h after treatment with vehicle or Vitamin AD injectable, and pregnancy was assessed 39 days post-AI by transrectal palpation and ultrasonography (Created with Biorender.com).

Figure 3.

Effect of exogenous vitamin AD injectable on serum and follicular fluid concentrations of calcidiol/25 hydroxyvitamin D₃ (25OHD). These treatments were administered over three sequential estrous cycles between March to May in Corvallis, OR, USA. (A) Serum concentrations of 25OHD (mean ± SEM; ng/mL) of cows in control and vitamin D-treated (Vit D) synchronized cycles. Day −1 denotes the day of injection (hatched bars), day 0 is 24 h after injection (solid bars). Effect of synchronized cycle, P = 0.003; interaction effect cycle by cycle day, P = 0.38. Different uppercase letters indicate difference between cycles P < 0.05, there were no detectable differences between cycle day (day −1 vs. 0; both P > 0.05). (B) Follicular fluid concentrations of 25OHD (mean± SEM; ng/ml) in periovulatory follicles aspirated 24 h after vehicle (control, grey) or vitamin AD (Vit D, black) injection during these same synchronized cycles. Effect of treatment/cycle P = 0.04. Different uppercase letters indicate difference between cycles P < 0.05.

Figure 4.

Effect of exogenous vitamin AD injectable on serum concentrations of calcidiol/25 hydroxyvitamin D₃ (25OHD) in the fertility trial. These treatments were administered between April and June in Monroe, OR, USA. Serum concentration of 25OHD (mean ± SEM; ng/mL) of cows who received control and vitamin AD (Vit D) injections during synchronized cycles. Day −1 denotes the day of injection (hatched bars), day 0 is 24 h after injection (solid bars). Effect of treatment P = 0.059, effect of treatment by day of cycle P = 0.11. Asterisk (*) indicates difference between day −1 and day 0 within Vit D cycles P = 0.01.