Cows enrolled in a pilot randomized clinical trial evaluating pegbovigrastim administered to dairy cows at dry-off had reduced incidence of intramammary infection

Juliana Leite de Campos Department of Animal Science, Michigan State University, East Lansing, MI

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Jaimie M. Strickland Department Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI

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Jeff C. Gandy Department Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI

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Cara I. Robison Department of Animal Science, Michigan State University, East Lansing, MI

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Pamela L. Ruegg Department Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI

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Abstract

OBJECTIVE

The objective of this pilot study was to determine if an alternative dosing schedule of pegbovigrastim (PEG; Imrestor; Elanco Animal Health) affects mammary gland health, rear udder width, or milk production of healthy dairy cows.

ANIMALS

20 pregnant late-lactation Holstein cows in November 2019 through April 2020.

PROCEDURES

Cows were randomly assigned to receive subcutaneous injections with either 15 mg of PEG (PEG group; n = 10) or a sham injection with saline (0.9% NaCl) solution (control group; 10) administered 7 days before dry-off and at dry-off. Quarter milk samples were collected for bacterial culture and somatic cells before and after dry-off and after calving. Mammary gland width was assessed before and after dry-off. Daily milk yields were evaluated after calving.

RESULTS

The incidence of intramammary infection was 5 times greater for quarters of cows in the control group than for quarters of cows that received PEG. The effect of treatment on somatic cell count was not significant, but the effects of period and a treatment-by-period interaction were identified. Treatment did not significantly affect milk production in the subsequent lactation, but the effects of period and an interaction of treatment by period were identified. Rear udder width after dry-off was not significantly affected by treatment, but an effect of period was identified.

CLINICAL RELEVANCE

In this pilot study, cows treated with PEG using an alternative dosing schedule had reduced incidence of intramammary infection and an interaction of treatment by sampling period was observed for milk yield. These results suggest that further studies with larger numbers of cows are warranted.

Abstract

OBJECTIVE

The objective of this pilot study was to determine if an alternative dosing schedule of pegbovigrastim (PEG; Imrestor; Elanco Animal Health) affects mammary gland health, rear udder width, or milk production of healthy dairy cows.

ANIMALS

20 pregnant late-lactation Holstein cows in November 2019 through April 2020.

PROCEDURES

Cows were randomly assigned to receive subcutaneous injections with either 15 mg of PEG (PEG group; n = 10) or a sham injection with saline (0.9% NaCl) solution (control group; 10) administered 7 days before dry-off and at dry-off. Quarter milk samples were collected for bacterial culture and somatic cells before and after dry-off and after calving. Mammary gland width was assessed before and after dry-off. Daily milk yields were evaluated after calving.

RESULTS

The incidence of intramammary infection was 5 times greater for quarters of cows in the control group than for quarters of cows that received PEG. The effect of treatment on somatic cell count was not significant, but the effects of period and a treatment-by-period interaction were identified. Treatment did not significantly affect milk production in the subsequent lactation, but the effects of period and an interaction of treatment by period were identified. Rear udder width after dry-off was not significantly affected by treatment, but an effect of period was identified.

CLINICAL RELEVANCE

In this pilot study, cows treated with PEG using an alternative dosing schedule had reduced incidence of intramammary infection and an interaction of treatment by sampling period was observed for milk yield. These results suggest that further studies with larger numbers of cows are warranted.

The dry period marks the beginning of a critical transition for dairy cows and is marked by several physiologic and metabolic changes that help to determine future productivity, profitability, and longevity of dairy cows.1 Involution of the mammary gland after cessation of milking is an important physiologic transition that consists of distinct periods defined by active and steady involution, formation of colostrum, and initiation of lactation.2 Within 70 h after cessation of milking, the mammary gland accumulates about 70 to 80% of typical daily milk production and increased risk of intramammary infection (IMI) occurs, partly due to increased risk of milk leakage.24

In many dairy herds, 10 to 50% of mammary gland quarters have developed IMI by the end of a lactation,5,6 and these infections are usually treated with antimicrobials at dry-off. Blanket dry cow therapy (DCT; intramammary administration of antimicrobials to all quarters of all cows at dry-off) is used for treatment of infected quarters as well as prevention of new infections during the critical early dry-off period7 and has been routinely recommended as part of a mastitis control program.8,9 Blanket DCT is widely adopted by U.S. dairy farmers10 but contributes to increased use of antimicrobials on farms, often accounting for 11% to 48% of total antimicrobial usage.1114 As demonstrated by the continued reduction in bulk tank somatic cell count (SCC) observed in U.S. dairy herds,15 subclinical mastitis has steadily declined, decreasing the need for blanket DCT and favoring the usage of nonantimicrobial alternatives to prevent mastitis at dry-off.

Pegbovigrastim (PEG) is a 2-dose immunomodulator that is labeled to be given 7 days prior to calving and again at calving to enhance the immune system of dairy cows, with the overall objective of reducing the incidence of clinical mastitis during the early postpartum period. Pegbovigrastim is the active ingredient, which is a pegylated recombinant form of granulocyte colony-stimulating factor, which is a naturally occurring bovine cytokine.16 This cytokine stimulates production and differentiation of neutrophils by progenitor cells in bone marrow.16 Neutrophils are the primary defense against IMI, but while an increase in circulating neutrophils has been consistently reported for animals treated with PEG,1618 the apparent impact of this product on reducing clinical mastitis was not sufficient to maintain commercial sales in the United States and the product is not currently marketed. During the early dry period, polymorphonuclear cells and macrophages are the primary leukocytes in the mammary gland responsible for resorption of milk components, removal of degenerated epithelial cells, and facilitation of involution.19,20 Altering the dosing schedule of this product to coincide with dry-off (rather than calving) may result in faster involution and reduced need for antimicrobials. The modified administration schedule of PEG at dry-off is an innovative approach that could reduce antimicrobial usage while maintaining mammary gland health. We hypothesized that PEG may be an accelerator of mammary gland involution, and administration of this product at dry-off may decrease the risk of IMI in the subsequent lactation. The objective of this pilot project was to generate preliminary data on the effects of an altered administration schedule of PEG on selected clinical measurements of mammary gland health in healthy cows during the early dry and subsequent postpartum periods.

Material and Methods

Study design and herd characteristics

A pilot, negatively controlled, randomized clinical trial was conducted at the Michigan State University Dairy Cattle Teaching and Research Center (East Lansing, MI) from November 2019 to April 2020. The dairy farm milked approximately 200 lactating cows that were housed in tie stalls bedded with sawdust. Dry cows that were enrolled in this study were housed in tie stalls containing mattresses that were bedded daily with wood shavings. Cows were moved to a maternity pen before calving and remained there until calving. Cows were milked twice daily in a milking parlor that included automated daily milk yield recording. Animal health records were recorded in computerized dairy software (Dairy Comp 305; Valley Agricultural Software). Gram-negative core antigen (Bovilis J-5; Merck) vaccines were given to cows 2 weeks after dry-off, 2 weeks before the due date, and 4 weeks after calving (ACALV). Experimental procedures were approved by the Institutional Animal Care and Use Committee at Michigan State University (protocol No. 201900347).

Enrollment criteria

Pregnant late-lactation Holstein dairy cows (n = 20) that were finishing their first to fourth lactations and had body condition scores between 3.0 and 4.0 (using a scale from 1 [severe underconditioning] to 5 [severe overconditioning]) were enrolled based on the absence of clinical mastitis 30 days prior to enrollment and current composite SCC < 200,000 cells/mL. Due to the high prevalence of bovine leukemia virus in this herd, seropositive cows were enrolled only if a blood count demonstrated < 10,000 lymphocytes/mL at the time of enrollment.

Intervention

Cows were blocked by parity (1, 2, and ≥ 3), bovine leukemia virus status (seronegative, seropositive), and milk yield (low ≤ median 305-day mature equivalent; high > median 305-day mature equivalent) using a statistical program.21 High-producing cows were defined based on milk yield greater than median milk production of cows enrolled in the study. Subcutaneous injections were administered in the morning after collection of milk samples. Cows assigned to the treatment group received 15 mg of PEG (2.7-mL prepackaged dose) 7 days before dry-off (BDRY) and a second dose on the day of dry-off (DRY). Cows assigned to the control group received a 2.7-mL sham injection of 0.9% saline (NaCl) solution using the same schedule. All treatments were administered by one researcher (JCG), and other researchers responsible for sample collection and analysis were blinded until initial data analysis was completed. Cows were dried off using only an iodine postdip (EDX-1000; Proactive Solutions), and no intramammary antimicrobials or internal teat sealants were given at DRY. An external teat sealant (T-Hexx Dry; Huvepharma) was applied to all quarters after the last dry period milk sample was collected 14 days after dry-off (ADRY).

Collection of clinical data

Rear udder width

Physical changes in mammary gland dimensions were assessed once after morning milking by measuring the distance between marks made on the rear left and right mammary gland similar to Larsen et al.22 Mammary glands were marked with a permanent marker in the center of each rear gland, and marks were reapplied as necessary. Manual measurements were taken from the middle right rear mammary gland to the middle left rear mammary gland following the curve of the udder. Data were collected at defined intervals before BDRY, DRY, and ADRY (Figure 1).

Figure 1
Figure 1

Sampling schedule for the outcomes of interest in a randomized clinical trial evaluating the clinical effects of pegbovigrastim (15 mg, SC; PEG group, n = 10) versus sterile saline (0.9% NaCl) solution (2.7 mL, SC; control group, 10) administered to dairy cows 7 days before dry-off (BDRY) and on the day of dry-off (DRY) between November 2019 and April 2020. Data collection occurred BDRY, at DRY, after dry-off (ADRY), and after calving (ACALV).

Citation: American Journal of Veterinary Research 84, 1; 10.2460/ajvr.22.08.0130

Milk samples

Quarter milk samples or secretions were collected in the morning on days 7 and 2 BDRY, at DRY, on days 7 and 14 ADRY, and on days 5, 10, and 14 ACALV and used for microbiological culture and determination of SCC (Figure 1). Milk samples were collected either during the first milking of the day (on days 7 and 2 BDRY, at DRY, and on days 5, 10, and 14 ACALV) or in the dry pen (on days 7 and 14 ADRY). Milk samples for microbiological analysis were aseptically collected following National Mastitis Council guidelines23 immediately stored on ice until they were transported to the laboratory. After collecting samples during the dry period, an iodine post-dip was applied to all quarters and on day 14 ADRY, an external teat sealant was administered to all quarters. Milk samples were immediately frozen at −20°C upon arrival at the laboratory.

Microbiological analysis

Initial microbiological analysis was performed at Michigan State University following National Mastitis Council guidelines.23 Milk samples were thawed at room temperature and 10 μL of milk were plated onto one-quarter of a trypticase soy agar plate that contained 5% blood agar and 0.1% esculin (Thermo Fisher Scientific). An additional 10 μL of milk was inoculated onto one-quarter of a MacConkey agar plate (Laboratory for Udder Health, University of Minnesota). Plates were incubated at 37°C for 48 h. Microbiological diagnosis was defined at the quarter level and an IMI was defined as the isolation of ≥ 300 CFU/mL of identical colonies. Milk samples were considered contaminated when 3 or more different colony types were isolated from the same sample. After growth on primary agar, species-level identification of colonies was performed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry at the Michigan State University Veterinary Diagnostic Laboratory. A new IMI was defined as identification of a different pathogen in the same quarter within 14 days or in the first sample collected ACALV. Recurrent IMI was defined as identification of the same pathogen in milk from the same quarter within 14 days or in the first sample collected ACALV. Clinical mastitis was defined as the presence of abnormal milk regardless of other clinical signs. Somatic cell count of quarter milk samples was determined using a commercially available automated counting system (Direct Cell Counter; Delaval).

Statistical analysis

Analyses were performed using statistical software (SAS version 9.4; SAS Institute Inc). Somatic cell count was log10 transformed to achieve normality, and mean, SE, minimum, and maximum were used to describe parity, days in milk (DIM), number of days dry, log10 SCC, and milk yield (kg).24 Univariant analyses were used to assess potential differences between treatment groups and parity, DIM at DRY, days dry, log10 SCC at DRY, and milk yield (kg) at DRY.25 Outcome variables were defined as incidence of IMI, log10 SCC, udder width, and milk production. Fisher exact test and odds ratio were performed to assess the difference in IMI between treatment groups.26 Data from quarters with IMI at day 7 BDRY were not used to calculate incidence of IMI.

Quarter-level SCC values were averaged for each cow and assessed at the cow level. The effect of treatment on SCC was determined in a repeated measure analysis with SCC as the dependent variable.27 Independent variables included treatment (control, PEG), time (on day 2 BDRY, at DRY, on days 7 and 14 ADRY, and on days 5, 10, and 14 ACALV), and the interaction of treatment and time, cow (treatment) was included as a random effect. Somatic cell count on day 7 BDRY was included as covariate for analysis of SCC as treatment was administered after samples were collected.

The effect of treatment on milk production was determined in a repeated measure analysis with milk production as the dependent variable.27 Independent variables included treatment (control; PEG), time (days 1 and 2 BDRY and days 5, 10, 14, 30, 60, and 120 ACALV), and the interaction of treatment and time, with cow (treatment) as a random effect. Milk yield on day 7 BDRY was included as covariate as treatment was administered after samples were collected and due to potential difference in milk yield between treatment groups.

The difference in udder width for each rear mammary gland was calculated by dividing the rear udder measurements taken at each period by the measurements at day 7 BDRY. The effect of treatment on change in udder width was determined in a repeated measure analysis with the percent of change as the dependent variable.27 Independent variables included treatment (control, PEG), time (on days 1 and 2 BDRY, at DRY, and on days 1, 2, 4, 7, and 14 ADRY), and the interaction of treatment and time, with cow (treatment) as a random effect. The Bonferroni test for multiple comparisons was used to separate means for log10 SCC, milk production, and udder width. Statistical significance was defined at P < .05.

Results

Lactating cows (n = 20) ending their first to fourth lactations were enrolled in this pilot study. The SCC and rolling herd average milk production of enrolled cows at their final monthly dairy herd improvement test prior to dry-off were 92,750 ± 21,830 cells/mL and 13,078 ± 634 kg, respectively. Cows were dried at 323 ± 8.3 DIM and were dry for 58 ± 1.2 days. No differences in parity, DIM, number of days dry, SCC, or milk yield were found between treatment groups (P > .22; Table 1).

Table 1

Comparisons of results for parity, days in milk (DIM) at the time of dry-off (DRY), days dry, log10-transformed somatic cell count (SCC) at DRY, and milk yield (kg) at DRY for 20 Holstein dairy cows enrolled in a randomized clinical trial evaluating the clinical effects of pegbovigrastim (15 mg, SC; PEG group, n = 10) versus sterile saline (0.9% NaCl) solution (2.7 mL, SC; control group, 10) administered 7 days before DRY and on the day of DRY between November 2019 and April 2020.

Control group PEG group
Variable Mean SEM Range Mean SEM Range P value
Parity 1.7 0.3 1.0–4.0 1.9 0.3 1.0–4.0 0.64
DIM 322.7 10.6 302.0–408.0 324.6 13.3 302.0–439.0 0.91
Days dry 58.9 1.8 45.0–65.0 56.0 1.4 49.0–63.0 0.22
Log10 SCC (cells/mL) 4.7 0.1 4.1–5.3 4.9 0.2 4.1–5.6 0.33
Milk yield (kg/y) 12,910 866 9,459–16,968 13,246 971 9,364–19,682 0.80

A total of 640 quarter milk samples were collected across 8 sampling periods. Of milk samples collected from cows in the control group (n = 320), 292 (91.3%) samples were culture negative, 4 (1.3%) were contaminated, and 24 (7.4%) were culture positive. Among culture-positive samples, non-aureus staphylococci were the most common pathogen, accounting for 75% (18 samples from 6 quarters of 4 cows) of IMI, followed by Streptococcus spp (4 samples from 4 quarters of 1 cow), Aerococcus viridians (1 samples from 1 quarter of 1 cow), and Trueperella pyogenes (1 sample from 1 quarter of 1 cow) (Table 2). Non-aureus staphylococci were composed by Staphylococcus haemolyticus, Staphylococcus chromogenes, and Staphylococcus xylosis. Of these IMI, only 1 cow had clinical mastitis (caused by Streptococcus spp.) during the dry period and received DCT after the last precalving sample was collected. One quarter from a cow in the control group had an IMI caused by non-aureus staphylococci at day 7 BDRY and remained infected across all samplings. Nine quarters from 3 cows developed a new IMI during the dry period and 3 quarters from 2 cows developed a new IMI ACALV.

Table 2

Quarter-level incidence of intramammary infection (IMI) for cows in PEG group (n = 40 quarters) or control group (40 quarters) described in Table 1 on days 7 and 2 before dry-off (BDRY), at DRY, on days 7 and 14 after dry-off (ADRY), and on days 5, 10, and 14 after calving (ACALV). Contaminated samples were defined as the identification of ≥ 3 colony types in the same sample. Incidence was calculated as the number of new IMIs divided by the number of noninfected quarters. Any IMI identified at day 7 BDRY was not included in the analysis.

Control group PEG group
BDRY DRY ADRY ACALV BDRY DRY ADRY ACALV
Pathogen 7 2 0 7 14 5 10 14 Total 7 2 0 7 14 5 10 14 Total
No growth 39 38 39 33 31 36 38 38 292 37 38 38 36 34 39 38 37 297
Staphylococcus aureus 1 1 1 3
Non-aureus staphylococci 1 1 1 4 4 3 2 2 18 2 2 2 2 3 1 2 14
Environmental streptococci 4 4 -
Aerococcus viridans 1 1 1 1
Trueperella pyogenes 1 1
Total IMI 1 1 1 4 9 4 2 2 24 3 2 2 2 3 1 2 4 18
Contaminated 1 3 4 2 3 5
Number of new cases (incidence%) 3 (7.7) 6 (15.4) 3 (7.7) 12 (30.8) 1 (2.7) 1 (2.7) 1 (2.7) 3 (8.1)

Of milk samples collected from cows in the treatment group (n = 320), 297 (92.8%) samples were culture negative, 5 (1.6%) samples were contaminated, and 18 (5.6%) samples were culture positive. Among culture-positive samples, non-aureus staphylococci were the most common isolated pathogen, accounting for 77.7% (14 samples from 4 quarters of 3 cows) of IMI, followed by Staphylococcus aureus (3 samples from 1 quarter of 1 cow) and Aerococcus viridians (1 sample from 1 quarter of 1 cow) (Table 2). Three quarters (3 cows) already had an IMI at day 7 BDRY. Of these IMI, 2 were caused by non-aureus staphylococci (2 quarter of 2 cow) and 1 (1 quarter of 1 cow) was caused by Aerococcus viridians. Only 1 quarter (1 cow) developed a new IMI during the dry period and 2 quarters (2 cows) developed a new IMI caused after calving.

In quarters that were healthy at dry-off, the incidence of new IMI was greater for quarters in the control group (12/39 quarters; 30.8%) than for quarters in the treatment group (3/37 quarters; 8.1%) (Χ2 = 6.1; P = .01). Approximately 23% (9/39 quarters) of the quarters in the control group had a new case of IMI during the dry period (at days 7 and 14 ADRY) as compared to none of the quarters in the PEG group. Across all sampling periods, as compared to mammary gland quarters in the treated group, quarters in the control group had 5 times (95% confidence interval: 1.3, 19.7) greater odds of IMI.

Least square mean log10 SCC of milk samples collected across all sampling periods from cows were 4.59 ± 0.05 and 4.68 ± 0.05 cells/mL, for quarters in the control and treatment groups, respectively, and were not affected by treatment (P = .23). The log10 SCC varied by sampling period (P < .001) and was least at 14 DIM and greatest at day 7 ADRY (Figure 2). An interaction between treatment and sampling period was found (P = .01). Somatic cell counts from cows treated with PEG were 13% greater on day 2 BDRY and 11% greater at DRY as compared to milk samples from cows in the control group (P = .002).

Figure 2
Figure 2

Least square mean (LSM) ± SEM log10-transformed somatic cell count (SCC) from milk samples collected on day 2 BDRY, at DRY, on days 7 and 14 ADRY, and on days 5, 10, and 14 ACALV for the PEG group (dashed line and rectangles) versus control group (solid line and triangles) described in Figure 1. For each group and time point, the rectangle or triangle represents the LSM, and the whiskers represent the SEM. Data were analyzed using a repeated measures analysis including the effect of treatment, period, and interaction between treatment and time. d = Days.

Citation: American Journal of Veterinary Research 84, 1; 10.2460/ajvr.22.08.0130

Changes in udder width were not affected by treatment (P = .23) but were affected by period (P < .001; Figure 3). In comparison to udder width at day 7 BDRY, beginning at day 2 ADRY, udder width of control cows increased until day 7 while udder width of treated cows decreased. As compared to udder width at day 7 BDRY, mammary gland width increased approximately 12% on day 1 ADRY and subsequently reduced approximately 16% on day 14 ADRY (Figure 3).

Figure 3
Figure 3

Graph of the LSM ± SEM of the difference (%) in mammary gland width on days 7, 2, and 1 BDRY, at DRY, and on days 1, 2, 4, 7, and 14 ADRY for the PEG group (dashed line and rectangles) versus control group (solid line and triangles) described in Figure 1. See Figure 2 for the rest of the key.

Citation: American Journal of Veterinary Research 84, 1; 10.2460/ajvr.22.08.0130

Milk yield was associated with sampling period (P < .001) and increased during the early lactation observation period. Across all sampling periods, no difference in milk yield was observed based on treatment group (control = 41.5 kg; PEG = 43.6 kg; P = .20), but there was a significant interaction between treatment and sampling period (P = .001; Figure 4).

Figure 4
Figure 4

Graph of the LSM ± SEM daily milk production on days 2 and 1 BDRY and on days 5, 10, 14, 30, 60, and 120 ACALV for the PEG group (dashed line and rectangles) versus control group (solid line and triangles) described in Figure 1. See Figure 2 for the rest of the key.

Citation: American Journal of Veterinary Research 84, 1; 10.2460/ajvr.22.08.0130

Discussion

It is well established that cows are most susceptible to new IMI during early dry and peripartum periods,2 due to increased risk of milk leakage, impairment of neutrophils,1 and persistence of IMI acquired during the previous lactation.2,6,28 Cows are more resistant to new IMI when the mammary gland reaches a steady involution phase.2 Thus, acceleration of mammary gland involution could help reduce risks of IMI during the dry and subsequent peripartum periods. While this study did not directly measure mammary gland involution, the potential effects of PEG administration on clinical measures of mammary gland health were evaluated. Administration of PEG is known to stimulate production and mobilization of neutrophils and may be a mechanism to speed mammary gland involution, therefore reducing the risk of IMI during this critical period. If effective, the novel approach of altering the administration schedule for PEG could reduce the need for administration of antimicrobials as well as provide a dosing schedule that coordinates with normal handling periods.

We evaluated mammary gland health based on the incidence of IMI during the dry period and after calving, as well as monitoring SCC during the same periods. Incidence of IMI was calculated at the quarter-level even though treatment was applied at the cow level, as quarters become independently infected. Although the prevalence of existing IMI before treatment (day 7 BDRY) was greater for quarters in cows that received PEG, the incidence of new IMI was greater in control quarters as compared to quarters of cows that received PEG. Of IMI cases in quarters of cows in the control group, approximately 75% occurred during the dry period as compared to no new IMI during the dry period of cows that received PEG. In both groups, the greatest proportion of IMI was caused by non-aureus staphylococci, which are commonly identified during this period.5,6 Neutrophils and macrophages are the primary cellular defense against intramammary infection, and the administration of PEG before dry-off might help prevent development of new IMI during the dry period and reduce the incidence of clinical mastitis cases after calving. Even with promising results, this study was designed as a pilot study to determine if future larger studies are warranted, and we enrolled only 10 cows per treatment. Results of the present study provided preliminary indication of efficacy that should be confirmed using more animals and in multiple herds.

While the approved PEG product is labeled for administration 2 weeks before calving, the impact of PEG on mammary gland health when administered during lactation has been evaluated.29 Powell et al29 treated 5 mid lactation cows with PEG, 7 days before challenging a single quarter with Escherichia coli. They reported that the concentration of neutrophils was almost 3 times greater than reported in previous studies that administered PEG during the peripartum period. In addition, they found that milk samples from cows treated with PEG had reduced numbers of bacteria in milk postchallenge and were protected from IMI. While we did not assess the number of neutrophils, the administration of PEG at dry-off may have resulted in greater concentration of neutrophils, thus providing additional resistance against IMI and explaining the difference in incidence of IMI between the treatment groups in this study.29

The transition from lactation to involution is marked by changes in udder size, structure, function, and composition of mammary gland secretion. Among methods used to evaluate mammary gland involution, udder measurement is an indirect, but practical method that has been previously used to assess this period. Changes in mammary gland dimensions were assessed to evaluate associations of reductions in feed intake, and milking frequency, with treatment of cabergoline at dry-off.22 They reported that change in udder engorgement was effectively assessed when interventions were used to modify the speed of dry-off.22 While we did not observe an overall impact of treatment on udder width, we were able to detect differences in udder width among periods, indicating that the method of measurement was sufficient to detect physiologic changes occurring during the early dry period.

After dry-off, increased SCC is expected as part of the normal physiologic process of involution.3,19 Polymorphonuclear cells and macrophages are the primary leukocytes in the mammary gland. After cows are dried off, these cells play an important role in resorption of milk components and in removal of degenerated epithelial cells.19,20 Thus, increased numbers of neutrophils stimulated by administration of PEG at dry-off may effectively speed mammary gland involution by facilitating removal of milk components and degenerate cells. Animals treated with PEG have been reported to maintain an increased number of neutrophils in blood relative to control animals for more than 7 days after treatment.16,18 Based on physiologic changes after cessation of milking, an increase in SCC is expected based on migration of neutrophils into the mammary gland. Across all sampling periods, we did not observe an effect of PEG on SCC, but this was expected, based on the expected duration of effect of treatment after administration of PEG. When the same model was used to test if SCC was affected by treatment only on day 2 BDRY and at DRY (days more likely to have an effect of treatment based on mode of action of this product), an effect of treatment on SCC was found, and PEG cows had a greater SCC than control cows (data not shown).

Surprisingly, as compared to the control cows, cows treated with PEG experienced a gradual increase in milk yield in the subsequent lactation that increased until 30 DIM and then persisted until 120 DIM. The significant interaction between treatment and sampling period was unexpected. After Bonferroni correction, we were unable to demonstrate differences in milk yield at individual periods, but as a pilot study, the data are intriguing and indicates that larger studies are potentially justified. Faster involution of milk secretory cells that result in enhanced productive capacity is a potential mechanism that warrants further investigation.2,30 Results from this pilot study indicate that use of an altered dosing schedule for PEG may warrant larger studies. Pilot studies are designed to generate data that indicate larger studies may be warranted and these results indicate that further studies that include more cows are necessary to understand the role of PEG on mammary gland cells renewal during dry period.

In conclusion, preliminary data from this pilot study indicated potential for use of PEG at dry-off to prevent new IMI and potentially speed mammary gland involution. As compared to the label schedule, administration of PEG during this period aligns with normal management schedules. In this pilot study, we observed that cows treated with PEG at dry-off had reduced incidence of IMI during dry period and after calving while maintaining low SCC and high milk yield in the subsequent lactation.

Acknowledgments

Funding and the PEG product (Imrestor) were provided by Elanco Animal Health, but Elanco Animal Health did not have involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.

We are grateful for the contributions of Dr. Lorraine Sordillo (deceased) who designed the overall study from which this work originated.

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  • Figure 1

    Sampling schedule for the outcomes of interest in a randomized clinical trial evaluating the clinical effects of pegbovigrastim (15 mg, SC; PEG group, n = 10) versus sterile saline (0.9% NaCl) solution (2.7 mL, SC; control group, 10) administered to dairy cows 7 days before dry-off (BDRY) and on the day of dry-off (DRY) between November 2019 and April 2020. Data collection occurred BDRY, at DRY, after dry-off (ADRY), and after calving (ACALV).

  • Figure 2

    Least square mean (LSM) ± SEM log10-transformed somatic cell count (SCC) from milk samples collected on day 2 BDRY, at DRY, on days 7 and 14 ADRY, and on days 5, 10, and 14 ACALV for the PEG group (dashed line and rectangles) versus control group (solid line and triangles) described in Figure 1. For each group and time point, the rectangle or triangle represents the LSM, and the whiskers represent the SEM. Data were analyzed using a repeated measures analysis including the effect of treatment, period, and interaction between treatment and time. d = Days.

  • Figure 3

    Graph of the LSM ± SEM of the difference (%) in mammary gland width on days 7, 2, and 1 BDRY, at DRY, and on days 1, 2, 4, 7, and 14 ADRY for the PEG group (dashed line and rectangles) versus control group (solid line and triangles) described in Figure 1. See Figure 2 for the rest of the key.

  • Figure 4

    Graph of the LSM ± SEM daily milk production on days 2 and 1 BDRY and on days 5, 10, 14, 30, 60, and 120 ACALV for the PEG group (dashed line and rectangles) versus control group (solid line and triangles) described in Figure 1. See Figure 2 for the rest of the key.

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    • Search Google Scholar
    • Export Citation
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    Zinicola M, Korzec H, Teixeira AGV, et al. Effects of pegbovigrastim administration on periparturient diseases, milk production, and reproductive performance of Holstein cows. J Dairy Sci. 2018;101(12):1119911217. doi:10.3168/jds.2018-14869

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    • Export Citation
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    Sordillo LM, Nickerson SC, Akers RM, Oliver SP. Secretion composition during bovine mammary involution and the relationship with mastitis. Int J Biochem. 1987;19(12):11651172. doi:10.1016/0020-711X(87)90098-X

    • PubMed
    • Search Google Scholar
    • Export Citation
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    Zhao X, Ponchon B, Lanctot S, Lacasse P. Invited review: accelerating mammary gland involution after drying-off in dairy cattle. J Dairy Sci. 2019;102(8):67016717. doi:10.3168/jds.2019-16377

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    • Search Google Scholar
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    Larsen M, Franchi GA, Herskin MS, et al. Effects of feeding level, milking frequency, and single injection of cabergoline on feed intake, milk yield, milk leakage, and clinical udder characteristics during dry-off in dairy cows. J Dairy Sci. 2021;104(10):1110811125. doi:10.3168/jds.2021-20289

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    • Search Google Scholar
    • Export Citation
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    Powell EJ, Reinhardt TA, Casas E, Lippolis JD. The effect of pegylated granulocyte colony-stimulating factor treatment prior to experimental mastitis in lactating Holsteins. J Dairy Sci. 2018;101(9):81828193. doi:10.3168/jds.2018-14550

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 30.

    Capuco AV, Ellis SE, Hale SA, et al. Lactation persistency: insights from mammary cell proliferation studies. J Anim Sci. 2003;81(suppl 3):1831. doi:10.2527/2003.81suppl_318x

    • PubMed
    • Search Google Scholar
    • Export Citation

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