Table 4.

Least squares means and associated pooled SEM for immunohistochemical markers of mammary gland tissue harvested from Holstein cows in different environmental treatments (Env): pair-fed thermoneutral (PFTN) or heat stress challenge (HSC), and fed different dietary treatments as top-dress (Diet): rumen-protected Met (RPM) or no RPM (CON)

	Phase 21									P-value2
Variable	Env		Diet		PFTN		HSC		SEM	Env	Diet	Env × Diet
	PFTN	HSC	CON	RPM	CON	RPM	CON	RPM
Apoptotic cells, % of total cells3,4	1.3	2.4	2.0	1.8	0.7a	2.0b	3.3b	1.5ab	0.9	0.88	0.15	0.03
Proliferating cells, % of total cells4,5	0.4	0.4	0.3	0.5	0.3	0.6	0.3	0.4	0.1	0.40	0.39	0.34
Apoptotic cells:Proliferating cells4	5.4	8.6	7.9	6.0	2.6a	8.2bc	13.2c	3.9ab	3.6	0.91	0.33	0.005
MIST16	1,100	1,500	900	1,700	900	1,400	1,000	2,000	594	0.55	0.13	0.20
XBP16	230	1,150	560	810	110	340	1,010	1,280	408	0.01	0.18	0.10

¹During phase 2 of periods 1 and 2, environmental treatments were pair-fed thermoneutral (PFTN) and heat stress challenge (HSC), and dietary treatments were rumen-protected Met included with top-dress (RPM) or no rumen-protected Met included with top-dress (CON). Top-dress vehicle was 300 g of dried molasses.

²No significant sequence or period effect present (P > 0.10).

³Apoptotic cells = [total area of apoptotic mammary cells/total area of mammary cells] × 100.

⁴PFTN (n = 23), HSC (n = 25), CON (n = 24), RPM (n = 24), PFTN/RPM (n = 11), HS/RPM (n = 13), PFTN/CON (n = 12), HS/CON (n = 12).

⁵Proliferating cells = [total area of Ki-67 antigen expressing mammary cells/total area of mammary cells] × 100.

⁶Number of mammary tissue samples used for the analyses are as follows: PFTN (n = 19), HSC (n = 20), CON (n = 19), RPM (n = 20), PFTN/RPM (n = 10), HS/RPM (n = 10), PFTN/CON (n = 9), HS/CON (n = 10).