Mineral supplementation (injectable) improved reproductive performance in Holstein cows managed in a warm summer environment
Funding information
This project was funded by the Program of Development and Research Support—PROFAPI from the Instituto Tecnológico de Sonora (Ciudad Obregón, Sonora, México); Grant number: PROFAPI_2019_0047
Abstract
Sustainability of dairy production depends largely on reproductive efficiency that is affected by heat stress due to high ambient temperature and humidity during summer. Supplementation of minerals has been proposed as a management strategy to minimize adverse impact of heat stress on fertility. The objective of this study was to determine the effects of an injectable mineral supplement (Fosfosan) containing selenium (Se), copper (Cu), potassium (K), magnesium (Mg) and phosphorus (P) on the ovarian structures, reproductive hormones and conception rate of heat-stressed Holstein cows. Sixteen cows were assigned during summer to one of two treatments, a control group (CON; n = 8) and a mineral-supplemented group (SUP; n = 8). Ambient temperature and relative humidity records were collected and processed to estimate the temperature–humidity index (THI), which confirmed a heat-stressed environment during the study (avg. THI = 79.4). Cows were subjected to a fixed-time artificial insemination (FTAI) program using the CIDR-Synch synchronization protocol. Traits indicative of ovarian activity were recorded during and after this protocol, as well as serum concentrations of reproductive hormones. Pregnancy diagnosis was made 28 and 35 d after FTAI. A completely randomized block design with repeated measures over time was performed to study ovarian functional structures and its hormonal profiles. Correlations and regressions were estimated to study relationships between ovarian structures and related hormones. Mineral supplementation did not increase follicular diameter or follicular populations (p > .05), yet tended to increase corpus luteum diameter (p < .10), and it enhanced (p < .01) oestrogen and progesterone serum concentrations and improved (p < .05) cow's conception rate. Diameter of dominant follicles and corpus luteum was correlated (p < .05) with oestrogen and progesterone levels, respectively, but only in mineral-treated cows. Two additional dairy herds were evaluated to confirm that mineral supplementation improved conception rate during the heat stress period (objective 2). Cows from dairy 1 received FTAI during winter (n = 401) and summer (n = 240), whereas cows from dairy 2 were bred after natural detected oestrus during winter (n = 558) and summer (n = 314). Conception rates were higher (p < .05) in winter than summer and they improved (p < .05) with mineral supplementation, but only in cows managed during summer. In conclusion, supplementation of minerals enhanced hormonal secretion from ovarian structures and improved conception rate in Holstein cows exposed to summer heat stress.
1 INTRODUCTION
Reproductive management in Holstein cows is a complex process affected by several factors, and its efficiency largely impacts genetic improvement, total milk production and financial sustainability of dairy herds (Muller et al., 2018). A major factor influencing fertility in cows managed in semi-arid regions is heat stress, which disrupts reproductive processes leading to a pronounced depression in conception rate (Wolfenson & Roth, 2019).
Lactating dairy cows are sensitive to heat stress due to their accelerated metabolic status, as heat production exceeds body heat loss and increases internal temperature (Polsky & von Keyserlingk, 2017). As ambient heat approaches body temperature, pathways of heat removal by animals such as convection, conduction and radiation become inefficient, with evaporation being the only functional strategy to dissipate heat. However, when the ambient humidity rises, the evaporative functions of thermal regulation such as panting and sweating become limited (Berman, 2006). Under such heat stress conditions, the difficulty to dissipate internal heat load to the environment is responsible for impaired reproduction (Wolfenson & Roth, 2019).
Effects of heat stress on reproduction can be reflected through the oestrous cycle, as this thermal effect interferes with follicular growth and corpus luteum development (Schüller et al., 2017). As a result, there is a reduction in the secretion of reproductive hormones such as LH, oestrogens (E2) and progesterone (P4), leading to alterations in the animal's reproductive behaviour and subsequent fertility (Aguiar et al., 2020). Also, hyperthermia from exposure to heat stress elevates ovarian and uterine temperatures affecting the development of oocytes and embryos, which compromises fertilization and embryo survival rates (Hansen, 2019).
Mineral supplementation has been proposed as a management strategy to counteract negative effects of heat stress (Kumar, 2015). Providing minerals improved respiration rate and rectal temperature, physiological indicators of heat stress, and enhanced milk composition and quality (Del Río-Avilés et al., 2021). Minerals are also involved in the synthesis and function of enzymes that protect the body from damages caused by heat stress (Khorsandi et al., 2016). In addition, minerals appear to be needed to support structure and function of granulosa and theca cells promoting ovarian follicular and luteal growth (Ceko et al., 2015).
Objective herein was to evaluate the effects of parenteral administration of minerals such as selenium, copper, potassium, magnesium and phosphorus on the ovarian structures, steroid reproductive hormones and conception rate in heat-stressed Holstein cows. The second objective was to validate the influence of mineral supplementation on conception rate in two additional commercial dairies.
2 MATERIALS AND METHODS
The ethics committee of the Instituto Tecnológico de Sonora (ITSON) approved all procedures and animal care conditions performed in this study.
2.1 Experimental location
This research was conducted from 11 June to 24 July 2018 at the Academic Unit of Dairy Production of the Instituto Tecnológico de Sonora, located in Block 910 of the Yaqui Valley, México. The geographic coordinates are 27°21′ North Latitude and 109°54′ West Longitude. The site has an altitude of 46 m over sea level and the climatic conditions vary from dry to semi-humid, with summer rainfall, average annual precipitation of 371.6 mm and average annual temperature of 23°C. Ambient temperature up to 48°C and THI up to 85 units can be recorded in this zone during the summer (Leyva-Corona et al., 2018).
2.2 Animals and experimental design
The study included an initial population of Holstein cows (n = 16) with average body weight of 602 ± 32.4 kg, 5–6 years old, 3–4 lactations, averaging 128 ± 8.6 days in milk and 25.76 ± 1.43 L of milk/day, and body condition score between 2.5 and 3.0 (where 1 is emaciated and 5 is obese). The experimental units were assigned under a randomized complete block design to one of two treatments, and number of lactations served as blocking factor. Treatments were as follows: (1) control cows (CON; n = 8), which did not receive any supplement with minerals, and (2) mineral-supplemented cows (SUP; n = 8), which received 3 intramuscular injections (10 ml each) of a mineral supplement (Fosfosan®, Virbac Lab Santa Elena; Table 1). The first application was made at the beginning of the experiment (Day −18). The second application was made 8 days later, at the beginning of the ovulation synchronization protocol ‘CIDR-Synch’ (Day −10). Finally, the third application was delivered at the time of FTAI (Day 0), as observed in Figure 1.
| Component | Quantity |
|---|---|
| Sodium glycerophosphate 5.5 H2O | 14.00 g |
| Monosodium phosphate 2 H2O | 20.10 g |
| Copper chloride 2 H2O | 0.40 g |
| Potassium chloride | 0.60 g |
| Magnesium chloride 6 H2O | 2.50 g |
| Sodium selenite | 0.24 g |
| Excipient | 100.00 ml |
The cows were fed a total mixed ration formulated with 85% forage (60% corn silage, 25% alfalfa hay) and 15% of commercial energy concentrate added with vitamins A, D, E and calcium to support milk production. The diet was formulated to meet the nutritional requirements of lactating Holstein cows weighing 600 kg and producing 28 kg of milk corrected for 3.5% fat (NRC, 2001). The chemical composition of the diet was 15.3% crude protein, 30.3% neutral detergent fibre and 1.2 Mcal/kg net lactation energy. Mineral content in the diet met the requirements for lactating dairy cows (Table 2). Cows were housed in pens (34 m by 22 m) with 8.5 m2 of shade per cow and had free access to water.
| Corn silage | Alfalfa hay | Concentrate | Requirement | |
|---|---|---|---|---|
| Calcium, % | 0.16 | 0.15 | 0.50 | 0.51 |
| Phosphorus, % | 0.16 | 0.14 | 0.37 | 0.33 |
| Magnesium, % | 0.10 | 0.08 | 0.18 | 0.20 |
| Potassium, % | 0.56 | 0.60 | 0.41 | 1.04 |
| Sodium, % | 0.14 | 0.21 | 0.33 | 0.23 |
| Copper, ppm | nd | nd | 12 | 10 |
| Selenium, ppm | 0.039 | 0.033 | 0.090 | 0.100 |
| Iron, ppm | 76 | 78 | 83 | 50 |
| Zinc, ppm | 18 | 22 | 48 | 40 |
2.3 Reproductive management
Cows were subjected to a trans-rectal ultrasonography inspection to evaluate the integrity of their reproductive tract using a 7.5 MHz transducer (Sonosite MicroMaxx TM®). Then, cows received an ovulation synchronization protocol, which consisted of an initial intramuscular (im) application of 0.01 mg of GnRH (Fertagyl®, Intervet) in conjunction with the placement of an intravaginal device for slow progesterone release (CIDR®, Pfizer) at Day −10. The CIDR was removed 7 days later (Day −3) followed by application of 25 mg im of prostaglandin F2α (PGF2α, Lutalyse®, Pfizer). Two days later, the cows received the second dose of 0.01 mg of GnRH (Day −1), and 24 hr later, they were inseminated at fixed time (FTAI; Day 0). Pregnancy diagnosis was performed at 28 and 35 days after FTAI using ultrasonography scanning. Conception rates were calculated for each treatment as the number of pregnant cows over total of FTAI services at Days 28 and 35. The difference in conception rate between Days 35 and 28 was considered as pregnancy loss.
2.4 Climatic variables
Ambient temperature (AT) and relative humidity (RH) data were recorded by a nearby weather station (Telemetery Gateway A840®, Adcon Telemetry Inc.) with the addVANTAGE 3.45® software package and sensors located 2 m above the ground. The meteorological station belongs to the Network of Automatic Meteorological Stations of Sonora (http://www.siafeson.com/remas). The THI was calculated using both AT and RH records following the formula THI = 0.81 AT + RH (AT − 14.4) + 46.4 (Mader et al., 2006).
2.5 Ovarian activity evaluation
Ovarian activity was evaluated using the following measurements: follicular population (FP), dominant follicle (DF) diameter, corpus luteum (CL) diameter and CL cavity diameter. These measurements were obtained using an ultrasonography machine equipped with a 7.5 MHz rectal transducer. The trait FP was determined on Day −7 of the synchronization protocol and Day 3 after FTAI, while diagnosis of DF diameter was performed on Days −3 and −2 of the protocol. In addition, CL and CL cavity diameters were measured on Day −3 of the protocol, as well as on Days 4, 9 and 19 after FTAI (Figure 2).
2.6 Blood samples and hormone measurements
Blood samples were collected from the initial population (n = 16) by puncture of the coccygeal vein using a BD vacutainer® blood sampling kit (blood collection needle measuring 0.8 × 38 mm, tube holder sleeve and 10 ml tubes without anticoagulant and with separating gel). The samples were maintained under laboratory conditions (18°C) and centrifuged at 3500 g for 20 min (Beckman coulter® centrifuge, model Allegra™ X-22). Then, serum was collected in Eppendorf tubes (2 ml) and used for hormone determinations.
Blood samples for P4 determinations were collected on Day −3 of the protocol and Days 4, 9 and 19 after FTAI, whereas samples for E2 measurements were collected on Days −3 and −2 of the CIDR-Synch protocol. Commercial ELISA kits were used to determine serum levels of both hormones (i.e. Bovine Progesterone ELISA Kit and Bovine Estrogen ELISA kit; MyBio Source LLC). The kit for P4 had a sensitivity of 0.2 ng/ml, and the intra-assay coefficient of variation was 11.3%. The kit for E2 had sensitivity up to 5 pg/ml with an intra-assay coefficient of variation of 8.9%.
2.7 Validation populations (commercial dairies)
Two additional dairy herds neighbouring the initial cattle population were tested during winter and summer periods using the same experimental design in early lactating Holstein cows, in order to validate the influence of mineral supplementation on fertility during heat stress.
The first test population (Dairy 1) included 401 lactating Holstein cows averaging 92.64 ± 1.32 days in milk and 27.65 ± 0.24 L of milk/day, which were randomly assigned to the two groups from January through April (i.e. cool period; THI = 54–71 units). These groups were mineral-supplemented cows (n = 205) that received Fosfosan injections (10 ml) before FTAI (Days −14 and −7) and during FTAI (Day 0), and non-supplemented control cows (n = 196). These cows started an oestrous synchronization regimen and received FTAI approximately 90 days post-calving. Then, pregnancy diagnosis was performed by ultrasound 28 d after FTAI. Similar management was performed from May through July (i.e. warm period, THI = 72–85 units), when lactating cows were also divided in mineral-supplemented (n = 112) and non-supplemented or control cows (n = 128).
The second test population (Dairy 2) involved 558 lactating Holstein cows averaging 92.14 ± 1.12 days in milk and 28.96 ± 0.21 milk litres/day. Cows were divided into mineral-supplemented (n = 291) and non-supplemented or control (n = 267) groups from December through March. Treated cows received a Fosfosan injection (10 ml) at Day 60 post-partum and during artificial insemination (AI), which was performed approximately 90 days post-partum after a natural detected oestrus. Control cows also received AI after natural oestrous in the same cool period. Pregnancy diagnosis was performed by ultrasound 28 days after AI. The same treatments were performed from May through August. Both mineral-supplemented (n = 146) and control (n = 168) cows were also subjected to AI after a natural oestrus and tested for pregnancy 28 days later.
Cows from dairies 1 and 2 experienced moderate-to-severe heat stress as they were exposed to a THI ranged from 72 to 85 units during the summer period (Zimbelman et al., 2009).
2.8 Statistical analyses
Descriptive statistics for climatic variables were estimated using PROC MEANS of SAS 9.3 statistical package (SAS Institute Inc., 2011). Reproductive variables were subjected to an analysis of variance using a randomized complete block design with repeated measurements over time using PROC MIXED. The number of lactations (two categories: three or four) was considered as a blocking factor. The model included fixed effects of treatment, block, time (day of measurement) and treatment by time interaction, as well as days in milk and body condition as linear covariates. The animal nested within each treatment served as random effect. Several variance–covariance structures were evaluated and the variance component (VC) structure showed the best fit according to AIC and BIC criteria. Means were separated with the PDIFF/LSMEANS option, considering as significant differences when alpha was <.05 and tendency when alpha was .05 > and <.10.
A correlation analyses was performed using PROC CORR to analyse associations (p < .05) between ovarian structures and serum levels of reproductive hormones within experimental groups. The PROC REG procedure was used to perform linear regression analyses.
Finally, the effects of treatment and season on conception rates were analysed by means of the Fisher's exact and chi-square tests using the PROC FREQ procedure.
3 RESULTS
3.1 Ovarian structures and hormone concentrations
Results of analyses of variance for ovarian structures (FP, DF diameter, CL diameter and CL cavity diameter) are presented in Table 3. An interaction effect between treatments and time was detected for P4 (p < .05). The treatment increased E2 concentrations (p < .0001) and was tendency for CL diameter (p < .10). In addition, both FP and CL cavity diameter increased across the time (p < .05).
| Trait | Treatments | p-Value for fixed effects | |||
|---|---|---|---|---|---|
| SUPa | CONb | Treatment | Time | Treat × time | |
| Follicular population (number) | 10.95 ± 1.64 | 9.86 ± 1.86 | .5417 | .0046 | .4015 |
| Dominant follicle diameter (mm) | 11.76 ± 0.97 | 11.69 ± 1.00 | .8583 | .6887 | .9462 |
| Corpus luteum diameter (cm) | 3.61 ± 0.67 | 1.52 ± 0.75 | .0567 | .2877 | .6702 |
| Corpus luteum cavity diameter (cm) | 0.46 ± 0.08 | 0.58 ± 0.09 | .3551 | .0216 | .3936 |
- a Mineral-supplemented cows.
- b Non-supplemented or control cows.
Concentrations of P4 between treatments for the different sampling days (time) are presented in Figure 3. Serum levels of P4 were similar (p > .05) between SUP and CON groups on Day −3 of the CIDR-Synch protocol (1.57 vs. 1.47 ng/ml, respectively) and Day 4 after FTAI (0.90 vs. 0. 56 ng/ml, respectively). However, a treatment effect (p < .01) was detected for P4 levels between SUP and CON groups on Day 9 (1.93 vs. 1.26 ng/ml, respectively) and Day 19 (1.44 vs. 0.55 ng/ml, respectively) after FTAI.
Oestradiol concentrations were greater (p < .01) in SUP versus CON group during Days −3 (5.95 vs. 4.85 pg/ml) and −2 of the CIDR-Synch protocol (6.49 vs. 5.02 pg/ml; Figure 4).
Correlations among ovarian structures and reproductive hormone concentrations are presented in Table 4. Diameter of DF was associated with E2 levels (r = .598; p < .05), whereas CL diameter was associated with P4 concentrations (r = .785; p < .05) in mineral-supplemented cows. In these females, CL diameter resulted as predictor for P4 levels (R2 = .62; p < .05; Figure 5a), and the regression equation (y = 0.89x − 0.25) suggested that P4 serum concentration would increase 0.89 ng/ml per unit of change in CL diameter. However, this associative relationship was lower and non-significant in control cows (R2 = 0.12; p > .05; Figure 5b).
| Trait | FP | DF | CL | CLC | E2 | P4 |
|---|---|---|---|---|---|---|
| Follicular population (FP) | – | .1402 | .1128 | .0914 | .2791 | .2621 |
| Dominant follicle (DF) | .1065 | – | .4165 | .1583 | .5982* | .3817 |
| Corpus luteum (CL) | .1207 | .2601 | – | −.3476 | .2751 | .7850* |
| Corpus luteum cavity (CLC) | .3146 | .0892 | −.1227 | – | .1639 | .1854 |
| Oestradiol (E2) | −.1668 | .2843 | −.3549 | .2601 | – | .1521 |
| Progesterone (P4) | .2325 | −.3004 | .3425 | .1338 | −.2738 | – |
- * and bold values are significance at p < .05.
3.2 Conception rates
Mineral-supplemented cows had higher (p < .05) conception rates compared with control cows at Days 28 (62.5 vs. 25.0%, respectively) and 35 (50 vs. 0%, respectively) after FTAI in the initial population, and showed a lower pregnancy loss (12.5 vs. 25.0%, respectively).
In cooperating commercial dairies, conception rates were higher (p < .05) in winter than in summer. However, in these validation herds, the conception rate improved (p < .05) in cows supplemented with minerals during summer, but not in winter (Table 5).
| Treatments | Summer season | Winter season | ||
|---|---|---|---|---|
| N | % (n) | N | % (n) | |
| Station 1 | ||||
| CON | 128 | 14.1a** (18) | 196 | 38.8a** (76) |
| SUP | 112 | 33.0b* (37) | 205 | 45.4a* (93) |
| Station 2 | ||||
| CON | 168 | 18.4a** (31) | 267 | 47.9a** (128) |
| SUP | 146 | 39.7b* (58) | 291 | 55.7a* (162) |
- a,bDifferent letters indicate significant difference (p < .05) between treatments according to ‘chi-square’ test.
- **Two asterisks indicate significant difference (p < .01) between seasons according to ‘chi-square’ test.
- *One asterisk indicates significant difference (p < .05) between seasons according to ‘chi-square’ test.
4 DISCUSSION
Ovarian components (i.e. follicles, oocytes and CL) in dairy cows are highly sensitive to adverse effects of heat stress (Wolfenson & Roth, 2019), which also alters the secretion patterns of reproductive hormones. Mineral supplementation appears to re-establish structural components and function of the ovaries from cows exposed to heat stress (Ceko et al., 2015). To address this issue, a commercial mineral supplement (Fosfosan) containing Se, Cu, Mg, K and P was injected to heat-stressed dairy cows to evaluate its effects on ovarian structures, concentration of reproductive hormones and conception rate. Similar to findings reported by Wolfenson and Roth (2019), we also observed a significant depression in E2 and P4 serum levels in heat-stressed cows. Although this effect was abated in FP, DF diameter and CL diameter in cows supplemented with minerals, they experienced an increased concentration of the reproductive hormones E2 and P4 suggesting an enhanced functionality of ovarian tissues from mineral supplementation. In addition, a closer relationship between ovarian structures and hormonal secretions was detected in mineral-treated cows.
To the best of our knowledge, this is the first report that mineral supplementation in Holstein cows exposed to sustained, long-term heat stress effects appeared to re-establish the ovarian function, and improved structural and functional relationships within the ovary. Ambient temperature and relative humidity created conditions of mild-moderate heat stress during June (avg. THI = 77.5 units); however, the climate became warmer and more humid during July leading to moderate-severe heat stress (avg. THI = 81.1 units; Zimbelman et al., 2009). Similar THI values have been previously reported during summer in the same region, affecting post-partum fertility traits in Holstein dairy cattle (Leyva-Corona et al., 2018).
Follicular populations (FP) were highly correlated with the total number of healthy follicles and oocytes (Mossa & Ireland, 2019). However, heat stress conditions altered follicular dynamic affecting FP and leading to a deficient reproductive behaviour (Wolfenson et al., 2000). This reduced fertility was also attributed to an increased number of atretic follicles and apoptotic granulosa cells (Li et al., 2016). Ovarian follicles are very sensitive to heat stress, and some weeks are required for follicular recovery (De Rensis et al., 2021).
In our study, FP appeared to be affected by heat stress and this follicular trait did not improve in mineral-supplemented cows. As reported by Van Emon et al. (2020), follicular cells did not appear to be influenced by mineral supplementation, which had no effect on number of follicles (Lamb et al., 2008), diameter of DF (González-Maldonado et al., 2017), follicular count and ovarian size in cows (Stokes et al., 2018).
In addition, heat stress induced an early emergence of the pre-ovulatory follicle leading to an extended duration of its dominance, which was associated with reduced fertility (Roth & Wolfenson, 2016). It could suggest that warm conditions affected functionality of the DF rather than growth, which agreed with findings from our study, as we did not observe any significant difference in follicular size between treatments.
In the current study, blood concentrations of E2 were higher in mineral-supplemented cows than those observed in the control group. The reduction in E2 levels in heat-stressed cows could be due to a decrease in gonadotropin receptor expression and a reduced aromatase activity in the granulosa cells (Li et al., 2016). This enzymatic reduction was associated with a deficient steroidogenic capacity of follicles under thermal stress leading to a decreased E2 synthesis in the dominant follicle (Wolfenson & Roth, 2019). Mineral supplementation improved both steroidogenesis and oocyte quality enhancing cattle reproductive performance (Van Emon et al., 2020).
The corpus luteum (CL) is a transient endocrine organ composed by small and large luteal cells, and its primary function is to secrete P4, an essential hormone required for maintenance of normal pregnancy in mammals (Wiltbank et al., 2012). In the current study, supplemented minerals in cows exposed to heat stress tended to increase CL size compared with control cows. Heat stress in cows appeared to disrupt the process of CL formation or the functionality of the CL, affecting P4 synthesis (Wolfenson & Roth, 2019). Supplementation with injectable trace minerals such as Se and Cu enhanced CL function and P4 secretion by increasing antioxidant enzymes, which control progesterone synthesis and release from the CL; however, CL size remained unchanged compared with a control group. These data suggested that after mineral supplementation, the CL appeared to produce P4 more efficiently, although did not influence CL growth (Van Emon et al., 2020).
The CL cavity diameter did not differ between SUP and CON groups (p > .05); however, it was influenced by the time or sampling day (p < .05). According to Perez-Marin (2009), the appearance of luteal cavities is associated with previous large pre-ovulatory follicles, although these luteal cavities did not appear to affect subsequent fertility. The finding of cavities within the CL has been reported through the oestrous cycle in Holstein females (Kastelic et al., 1990). Furthermore, the progress in the size of CL cavities depends on the growth and maturation of the CL (Kito et al., 1986). It could help to explain the increasing in the diameter of CL cavities, concomitant with CL maturation, that was observed in this study after the FTAI.
An interaction effect of treatment by day of sampling influenced serum concentrations of P4. Although all cows were exposed to sustained heat-stressed conditions during the study (THI >78 units), mineral supplementation increased P4 concentrations on Days 9 and 19 after FTAI compared with control cows. The significant decrease in P4 secretion observed in cows exposed to long-term, seasonal heat stress was attributed to a disruption in CL formation that affects the steroidogenic ability of the luteal cells (Wolfenson & Roth, 2019).
Supplementing Se increased plasma concentrations of P4 during late gestation in Holstein heifers (Kamada et al., 2014). Similarly, a significant increase in P4 concentrations was reported in heat-stressed sheep receiving a mineral supplement enriched with phosphorus (Senosy et al., 2018). Such a favourable effect of phosphorus could be attributed to its function in the phospholipids of the plasma membrane of luteal cells and its role in the synthesis of cyclic AMP in these cells.
In the current study, mineral-supplemented cows showed stronger correlations between ovarian structures and levels of P4 and E2, compared with heat-stressed control cows. These results suggested that heat stress altered the normal relationship existing between ovarian structures and their hormonal profiles probably due to a dysfunction in the ovary. The heat shock induced by exposure to an extremely warm climate could have increased reactive oxygen species (ROS), which affected growth and steroidogenic capacity of follicles and CL leading to an ovarian dysfunction (Roth & Wolfenson, 2016). Conversely, animals receiving supplementary minerals such as Se and Cu increased their antioxidant enzymatic activity, which improved follicle and CL growth, oocyte quality, and secretion of E2 and P4 helping to re-establish ovarian function (Van Emon et al., 2020). These reports can help to explain the predictive relationship between CL diameter and P4 levels observed in our study in mineral-injected cows, suggesting the functional recovery of the ovary.
However, the recovery of ovarian follicular structures seems to be delayed by a long-lasting effect of heat stress, which is more evident during the early growth phase of the follicular wave (Roth et al., 2001). Bovine oocytes collected from cows exposed to heat stress experienced a high incidence of disruption in maturation suggesting a compromised follicular physiology (Edwards et al., 2005). In addition, an altered biochemical composition within the follicular fluid has been reported in lactating dairy cows exposed to heat stress (Rispoli et al., 2019). These results support our findings because we did not observe a significant association between follicular population and E2 concentrations in supplemented cows, even though E2 levels appeared to be recovered from mineral supplementation.
Conception rate increased in mineral-supplemented cows exposed to heat stress compared with non-supplemented control groups. Moreover, conception rates in cooperative dairies 1 and 2 improved during winter compared with summer, with a significant effect between treatments observed in summer period. These results validated that mineral supplementation improved fertility in Holstein cows exposed to heats tress during summer in our study.
Heat shock severely affects the embryo development in lactating Holstein cows, which are very sensitive to heat effects (Hansen, 2019). Heat stress reduced weight, diameter and P4 secretion of the CL, altering the endometrial environment and leading to low fertilization rates (Fernandez-Novo et al., 2020). Thermo-tolerance appears to be associated with antioxidant protection. In vitro studies demonstrated a significant protection against heat shock in embryos treated with antioxidants such as anthocyanin (Sakatani et al., 2007). Mineral administration could be useful to ameliorate the negative effects of heat stress on conception rate in ruminants (Khorsandi et al., 2016; Krishnan et al., 2017). Supplementation of minerals (Fosfosal®, Virbac) improved conception rate versus a control group in Angus and Hereford cows (53.5% vs. 46.5%, respectively; Pessoa et al., 2017), and lactating Nelore cows (52.0% vs. 45.0%, respectively; Penteado et al., 2017), which were managed under a subtropical heat-stressed environment similar to that observed in the current study.
Finally, pregnancy loss from Weeks 4 to 5 of gestation seemed to decrease in heat-stressed cows receiving mineral supplementation. Heat stress is considered as a major factor impairing uterine environment and embryo development that may lead to an embryonic loss (De Rensis et al., 2021). However, even in these circumstances, the embryo utilizes minerals secreted by the uterine histotroph, which are essential for its growth and development (Perry et al., 2021). These results may help to explain the favourable effect of minerals supplemented in our study to reduce early pregnancy losses, which consequently improved conception rates.
5 CONCLUSIONS
Heat stress negatively influenced fertility in Holstein dairy cows by disrupting follicular dynamic and CL development, which reduced ovarian secretion of E2 and P4, and affected conception rate. An injected mineral supplement (Fosfosan) containing Se, Cu, K, Mg and P provided during summer appeared to re-establish ovarian functionality in heat-stressed lactating dairy cows. Such recovery improved relationships among ovarian structures and serum concentrations of E2 and P4, and increased conception rate. These results (i.e. increased cow conception rate) were validated in two large commercial dairies, evidencing the beneficial effect of mineral supplementation on fertility in heat-stressed dairy cattle.
ACKNOWLEDGEMENTS
We thank the Laboratorios Virbac of México for providing the mineral supplement tested in the study. Additionally, we would like to acknowledge the National Council for Science and Technology (CONACYT) of México for providing the student scholarship for the first author.
CONFLICT OF INTEREST
None of the authors have any conflict of interest to declare.
AUTHORS CONTRIBUTION
A.D. Del Río-Avilés designed and performed the experiment, and analysed data; A. Correa-Calderón, L. Avendaño-Reyes and U. Macías-Cruz designed the experiment and critically revised the manuscript. M.G. Thomas, R.M. Enns, and S.E. Speidel critically revised and edited the manuscript. M.A. Sánchez-Castro and R. Zamorano-Algandar collected and analysed data. P.A. López-Castro provided reagents and collected data. P. Luna-Nevárez designed and performed the experiment, provided reagents and materials, collected data and wrote the manuscript. All the authors read and accepted the manuscript.
ETHICAL APPROVAL
All procedures involving animals performed in this study were in accordance with the ethical standards of the Instituto Tecnológico de Sonora (ITSON) in which the study was conducted.
Open Research
DATA AVAILABILITY
The data used to support the findings of this study are available from the corresponding author upon reasonable request.
